KEX2 Cleavage regions of recombinant fusion proteins

ABSTRACT

The invention relates to a fusion DNA construct comprising a KEX2 region comprising a KEX2 site and a KEX2 site pre-sequence immediately 5′ to the KEX2 site, a fusion polypeptide, vectors and cells comprising the fusion DNA construct, methods for producing desired proteins from filamentous fungal cells and methods for enhancing the secretion and/or cleavage of a desired protein from a cell.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

Portions of this work were funded by Contract No. W911NF-05-C-0072 bythe Defense Advanced Research Projects Agency (DARPA) of the U.S.Accordingly, the United States Government may have certain rights inthis invention.

FIELD OF THE INVENTION

The present invention relates to increased secretion and cleavage ofdesired proteins, such as functional antibody proteins and industrialenzymes from filamentous fungi. The invention discloses fusion DNAconstructs, vectors and fusion polypeptides incorporating KEX2 regionsfor protein cleavage and methods of producing desired proteins.

BACKGROUND

During protein secretion in a fungal cell, certain proteins are cleavedby KEX2, a member of the KEX2 or “kexin” family of serine peptidase (EC3.4.21.61). KEX2 is a highly specific calcium-dependent endopeptidasethat cleaves the peptide bond that is immediately C-terminal to a pairof basic amino acids (i.e., the “KEX2 site”) in a protein substrateduring secretion of that protein. KEX2 proteins generally contain acysteine residue near the histidine residue of its active site and areinhibited byp-mercuribenzoate. The founding member of this group, theKEX2 peptidase of S. cerevisiae (Fuller et al., 1989, Proc. Natl. Acad.Sci. USA 86:1434-1438), cleaves the α-factor pheromone and killer toxinprecursors during their secretion.

Production of fusion polypeptides has been reported in a number oforganisms including E. coli, yeast and filamentous fungi. For example,bovine chymosin has been produced in Aspergillus niger as a fusion tofull length glucoamylase (GAI) (Ward et al., (1990) Bio/technology8:435-440; U.S. Pat. No. 6,265,204 and U.S. Pat. No. 6,590,078); humaninterleukin 6 (hIL6) has been produced in Aspergillus nidulans as afusion to full-length A. niger glucoamylase (GAI) (Contreras et al.,(1991) Biotechnology 9:378-381); hen egg white lysozyme (Jeenes et al.,(1993) FEMS Microbiol. Lett. 107:267-273) and human lactoferrin (Ward etal., (1995) Bio/Technology 13:498-503) have been produced in Aspergillusniger as a fusion to residues 1-498 of glucoamylase; and bovine chymosinhas been produced in Aspergillus niger as a fusion with full lengthnative alpha amylase (Korman et al., (1990) Curr. Genet. 17: 203-212)and in Aspergillus oryzae as a fusion with truncated forms of A. oryzaeglucoamylase (Tsuchiya et al., (1994) Biosci. Biotech. Biochem. 58:895-899). Reference is also made to Shoemaker et al., 1981Bio/Technology 1: 691-696; Nunberg et al., (1984) Mol. Cell. Biol.4:2306-2315 and Boel et al., (1984) EMBO J. 3:1097-1102. In some ofthese fusion proteins, a KEX2 protease recognition site (Lys-Arg) hasbeen inserted between a glucoamylase and a desired protein (e.g.,Contreras et al., 1991 and Ward et al., 1995). The inventors of thepresent invention have found that protein secretion and/or proteincleavage may be enhanced in a fusion protein when the KEX2 recognitionsite has been manipulated to include an amino acid KEX2 sitepre-sequence.

Specific literature of interest includes: Ward et al., (2004) Appl.Environ. Microbiol. 70:2567-2576; Goller et al., (1998) Appl. Environ.Microbiol. 64:3202-3208; La Grange et al., (1996) Appl. Environ.Microbiol. 62:1036-1044; Bergquist et al., (2002) Biochem. Biotechnol.100:165-176; Spencer et al., (1998) Eur. J. Biochem. 258:107-112;Jalving et al., (2000) Appl. Environ. Microbiol. 66:363-368); Brennerand Fuller (1992) Proc. Natl. Acad. Sci. 89:922-926; Durand et al.,(1999) Appl. Microbiol. Biotechnol. 52: 208-214; Ahn et al., (2004)Appl. Microbiol. Biotechnol. 64:833-839; Gouka et al., (1997) ApplMicrobiol Biotechnol. 47:1-11 Broekhuijsen et al., (1993) J. Biotechnol.31:135-145; MacKenzie et al., (1998) J. Biotechnol. 63:137-146 andpublished patent applications 20040018573 and 20050158825. Also U.S.Pat. No. 4,816,567 and U.S. Pat. No. 6,331,415 disclose processes forproducing immunoglobulin molecules in recombinant host cells. The abovecited literature is incorporated by reference herein for all purposes.

While numerous methods are available for the production of industrialenzymes and therapeutic proteins, there remains a need for alternativemethods of protein production and particularly for therapeutic proteinproduction, such as antibody production, which will result in relativelyquick scale up time and high levels of produced protein with limitedrisk of contamination by viral or other adventitious agents. The presentinvention answers this need.

SUMMARY OF THE INVENTION

A fusion DNA construct, vectors, a fusion polypeptide, a cell comprisingthe fusion DNA construct, and methods for enhancing the secretion and/orcleavage of a desired protein from a cell are provided. Morespecifically, a KEX2 region encompassed by the invention has beenincluded in a fusion polypeptide to provide for cleavage of a desiredprotein from the fusion polypeptide. Accordingly, the invention pertainsto a KEX2 region for protein cleavage.

In some embodiments, the invention relates to a fusion DNA constructencoding a fusion polypeptide, comprising in operable linkage from the5′ end of said construct, a promoter; a first DNA molecule encoding asignal sequence; a second DNA molecule encoding a carrier protein; athird DNA molecule encoding a KEX2 region, said KEX2 region comprising aKEX2 site and a KEX2 site pre-sequence immediately 5′ to the KEX2 site;and a fourth DNA molecule encoding a desired protein. In some aspects ofthis embodiment, the invention relates to a vector, such as anexpression vector, which comprises the fusion DNA construct, and inother aspects the invention relates to host cells transformed with thevector or comprising the fusion DNA construct.

In other embodiments, the invention relates to a fusion polypeptidecomprising from an amino terminus of said fusion polypeptide a firstamino acid sequence comprising a signal sequence functional as asecretory sequence; a second amino acid sequence comprising a carrierprotein; a third amino acid sequence comprising a KEX2 region, said KEX2region comprising a KEX2 site and a KEX2 site pre-sequence immediatelyN-terminal to said KEX2 site; and a fourth amino acid sequencecomprising a desired protein.

In further embodiments, the invention relates to a KEX2 region(X₄X₃X₂X₁B₁B₂) comprising a KEX2 site (B₁B₂) and a KEX2 sitepre-sequence (X₄X₃X₂X₁) immediately N-terminal to said KEX2 site.

In yet other embodiments, the invention relates to a process ofproducing a desired protein in a filamentous fungal host cell andparticularly in a Trichoderma cell, comprising obtaining a filamentousfungal host cell comprising a fusion DNA construct according to theinvention and culturing the filamentous fungal host cell under suitableconditions which allow the expression and secretion of the desiredprotein. In some aspects of this embodiment, the desired protein will berecovered. In other aspects of this embodiment, the cleavage of thedesired protein from the fusion polypeptide will be greater than thecleavage of the same desired protein from an equivalent fusionpolypeptide that lacks the KEX2 site pre-sequence. In other aspects ofthis embodiment, the secretion of the desired protein from the fusionpolypeptide will be greater than the secretion of the same desiredprotein from an equivalent fusion polypeptide, which lacks the KEX2 sitepre-sequence.

In an additional embodiment, the invention relates to a method foridentifying enhanced secretion and/or cleavage of a desired proteincomprising a) altering a KEX2 site pre-sequence of a parental fusionpolypeptide, said parental fusion polypeptide comprising a signalsequence; a KEX2 region comprising a KEX2 site and the KEX2 sitepre-sequence which is located immediately N-terminal to said KEX2 site,and an amino acid sequence comprising a desired protein to produce a setof test fusion polypeptides that are identical to said parental fusionpolypeptide except for said KEX2 site pre-sequence; b) evaluatingsecretion of said test fusion polypeptides and said parental fusionpolypeptide by a filamentous fungal cell; c) identifying a test fusionpolypeptide that has enhanced secretion and/or cleavage as compared tosaid parental fusion polypeptide.

In further aspects of this embodiment, the invention relates to a methodof identifying an optimized KEX2 site pre-sequence which comprises,testing a plurality of different test fusion polypeptides obtained asdescribed above; and determining which of said different test fusionpolypeptides has greater secretion and/or protein cleavage, wherein saidoptimized KEX2 site pre-sequence is the altered KEX2 site pre-sequenceof the test fusion polypeptide that has the greatest secretion and/orprotein cleavage.

BRIEF DESCRIPTION OF THE FIGURES

Certain aspects of the following detailed description are bestunderstood when read in conjunction with the accompanying drawings. Itis emphasized that, according to common practice, the various featuresof the drawings are not to-scale. On the contrary, the dimensions of thevarious features are arbitrarily expanded or reduced for clarity.Included in the drawings are the following figures:

FIG. 1 schematically illustrates an embodiment of a fusion polypeptideaccording to the invention, including a carrier protein, a KEX2 regionand a desired protein, wherein the carrier protein is illustrated as acellobiohydrolase I (CBH1) core/linker, which comprises the catalyticdomain and part of the linker region of the CBH1 protein and the desiredprotein is illustrated as an antibody light chain or heavy chain.

FIG. 2 depicts a map of the pTrex4-her2 light chain DNA2.0 plasmid usedfor the expression of a fusion polypeptide. The plasmid includes aTrichoderma reesei cbh1 promoter; a polynucleotide encoding a CBH1signal sequence and carrier protein; a KEX2 region inserted immediatelyafter the SpeI site, a polynucleotide encoding the desired proteinillustrated as an antibody (trastuzumab) light chain; a Trichodermareesei cellobiohydrolase (cbh1) terminator; an amdS Aspergillus nidulansacetamidase marker.

FIG. 3A-E provide the nucleotide sequence (SEQ ID NO: 103) (10885 bp) ofthe pTrex4-her2 light chain DNA2.0 plasmid of FIG. 2.

FIG. 4 shows a Western blot of supernatants of cultured Trichodermareesei cells comprising KEX2 region sequences as further described inexamples 1, 2, 3 and 4. Lane 1 represents a molecular weight marker (SeeBlue Plus 2, Invitrogen). Lanes 2 and 3 represent a GGGKR variant (SEQID NO: 5); lane 4 represents a GGGKRGGG variant (SEQ ID NO: 7); lane 5represents a VAVEKR variant (SEQ ID NO: 9) KEX2 region encompassed bythe invention; and lanes 6 and 7 represent a KRGGG variant (SEQ ID NO:2).

FIG. 5 shows a Western blot of supernatants of cultured Trichodermareesei cells comprising KEX2 regions encompassed by the invention asfurther described in example 5. Lane 1 represents a molecular weightmarker as described above. Lanes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and13 correspondingly represent VAVEKR (SEQ ID NO: 9), VAVWKR (SEQ ID NO:25), VAVGKR (SEQ ID NO: 26), VAVRKR (SEQ ID NO: 27), VAVTKR (SEQ ID NO:28), VAVVKR (SEQ ID NO: 29), VAVAKR (SEQ ID NO: 30), VAVLKR (SEQ ID NO:31), VAVDKR (SEQ ID NO: 32), VAVNKR (SEQ ID NO: 33), VAVYKR (SEQ ID NO:34), VAVHKR (SEQ ID NO: 35) KEX2 regions.

FIG. 6 shows a Western blot of supernatants of cultured Trichodermareesei cells containing KEX2 region sequences as further described inexamples 5, 6, and 7. Lanes 1 and 10 represent a molecular weightmarker, as described above. Lanes 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13,14, 15 and 16 correspondingly represent AAVEKR (SEQ ID NO: 38), GAVEKR(SEQ ID NO: 37), MAVEKR (SEQ ID NO: 36), LAVEKR (SEQ ID NO: 39), WAVEKR(SEQ ID NO: 40), KAVEKR (SEQ ID NO: 41), PAVEKR (SEQ ID NO: 42), DAVEKR(SEQ ID NO: 51), VAVEKR (SEQ ID NO: 9), HAVEKR (SEQ ID NO: 52), QAVEKR(SEQ ID NO: 47), SAVEKR (SEQ ID NO: 46), NVISKR (SEQ ID NO: 22), andSDVTKR (SEQ ID NO: 24) KEX2 regions.

FIG. 7 shows a Western blot of supernatants of cultured Trichodermareesei cells containing KEX2 region sequences as further described inexample 5. Lane 1 represents a molecular weight marker, as describedabove. Lanes 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11 correspondingly representVAVEKR (SEQ ID NO: 9), VGVEKR (SEQ ID NO: 56), VTVEKR (SEQ ID NO: 65),VEVEKR (SEQ ID NO: 55), VPVEKR (SEQ ID NO: 62), VWVEKR (SEQ ID NO: 67),VKVEKR (SEQ ID NO: 58), VRVEKR (SEQ ID NO: 63), VVVEKR (SEQ ID NO: 66),and VIVEKR (SEQ ID NO: 57) KEX2 regions.

FIG. 8 shows a Western blot of supernatants of cultured Trichodermareesei cells containing KEX2 region sequences as further described inexample 5. Lanes 1-11 correspondingly represent VADEKR (SEQ ID NO: 70),VAAEKR (SEQ ID NO: 69), VAFEKR (SEQ ID NO: 72), VAGEKR (SEQ ID NO: 73),VAIEKR (SEQ ID NO: 74), VANEKR (SEQ ID NO: 76), VALEKR (SEQ ID NO: 75),VASEKR (SEQ ID NO: 79), VAREKR (SEQ ID NO: 78) and VAPEKR (SEQ ID NO:83) KEX2 regions.

FIG. 9 shows an SDS-PAGE gel of supernatants of cultured A. niger cellscontaining a VAVEKR (SEQ ID NO: 9) KEX2 region as further described inexample 8. Lane 1 represents a molecular weight marker, Marker 12 MWstandard (Invitrogen). Lanes 2, 3, and 4 represent 3 transformants andcorrespond respectively to transformants A10, A11 and A12.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton, et al., DICTIONARYOF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, NewYork (1994), and Hale & Markham, THE HARPER COLLINS DICTIONARY OFBIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with thegeneral meaning of many of the terms used herein. Still, certain termsare defined below for the sake of clarity and ease of reference.

The term “recombinant” when used in reference to a cell, nucleic acid,protein or vector, indicates that the cell, nucleic acid, protein orvector, has been modified by the introduction of a heterologous nucleicacid or protein or the alteration of a native nucleic acid or protein,or that the cell is derived from a cell so modified. Thus, for example,recombinant cells express nucleic acids or polypeptides that are notfound within the native (non-recombinant) form of the cell or expressnative genes that are otherwise abnormally expressed, under expressed,over expressed or not expressed at all.

A “gene” refers to a DNA segment that is involved in producing apolypeptide and includes regions preceding and following the codingregions, e.g., the promoter and terminator, as well as interveningsequences (introns) between individual coding segments (exons).

The term “nucleic acid” encompasses DNA, RNA, single stranded or doublestranded and chemical modifications thereof. The terms “nucleic acid”and “polynucleotide” may be used interchangeably herein. Because thegenetic code is degenerate, more than one codon may be used to encode aparticular amino acid, and the present invention encompassespolynucleotides, which encode a particular amino acid sequence.

The term “DNA construct” means a DNA sequence which is operably linkedto a suitable control sequence capable of effecting expression of aprotein in a suitable host. Such control sequences may include apromoter to effect transcription, an optional operator sequence tocontrol transcription, a sequence encoding suitable ribosome bindingsites on the mRNA, enhancers and sequences which control termination oftranscription and translation.

The term “fusion DNA construct” or “fusion nucleic acid” refers to anucleic acid which comprises from 5′ to 3′ a number of polynucleotidesequences (e.g. a DNA molecule encoding a signal sequence, a DNAmolecule encoding a carrier protein, a DNA molecule coding for a KEX2site and a DNA molecule encoding a desired protein) operably linkedtogether and which encode a fusion polypeptide.

A “vector” refers to a polynucleotide sequence designed to introducenucleic acids into one or more cell types. Vectors include cloningvectors, expression vectors, shuttle vectors, plasmids, phage particles,cassettes and the like.

An “expression vector” refers to a vector that has the ability toincorporate and express heterologous DNA fragment in a foreign cell.Many prokaryotic and eukaryotic expression vectors are commerciallyavailable.

A “promoter” is a regulatory sequence that is involved in binding RNApolymerase to initiate transcription of a gene.

The term “signal sequence” refers to a sequence of amino acids at theamino terminus of a protein that directs the protein to the secretionsystem for secretion from a cell. The signal sequence is cleaved fromthe protein prior to secretion of the protein. In certain cases, asignal sequence may be referred to as a “signal peptide” or “leaderpeptide”. The definition of a signal sequence is a functional one. Themature form of the extracellular protein lacks the signal sequence whichis cleaved off during the secretion process.

“Under transcriptional control” is a term well understood in the artthat indicates that transcription of a polynucleotide sequence, usuallya DNA sequence, depends on its being operably linked to an element whichcontributes to the initiation of, or promotes transcription.

“Under translational control” is a term well understood in the art thatindicates a regulatory process which occurs after mRNA has been formed.

The term “operably linked” refers to juxtaposition wherein the elementsare in an arrangement allowing them to be functionally related. Forexample, a promoter is operably linked to a coding sequence if itcontrols the transcription of the sequence.

The term “selective marker” refers to a protein capable of expression ina host that allows for ease of selection of those hosts containing anintroduced nucleic acid or vector. Examples of selectable markersinclude but are not limited to antimicrobials (e.g., hygromycin,bleomycin, or chloramphenicol) and/or genes that confer a metabolicadvantage, such as a nutritional advantage on the host cell.

The terms “protein” and “polypeptide” are used interchangeably herein.The conventional one-letter or three-letter code for amino acid residuesis used herein.

The term “carrier protein” refers to a polypeptide sequence orfunctional portion thereof from a naturally secreted fungal polypeptide.

The term “antibody protein” used interchangeably with immunoglobulins(Igs), refers to a protein containing one or more polypeptides thatspecifically binds an antigen. Included by this term are antibodies ofany isotype, fragments of antibodies which retain specific binding toantigen, including, but not limited to, Fab, Fv, scFv, Fd, Fab′, Fv,F(ab′)₂ antibodies, antibody fragments that retain specific binding toantigen, monoclonal antibodies, chimeric antibodies, humanizedantibodies, single-chain antibodies, bi-functional (i.e. bi-specific)hybrid antibodies and fusion proteins comprising an antigen-bindingportion of an antibody and a non-antibody protein.

The monomeric form of an antibody comprises four polypeptide chains oftwo different types, one heavy and one light. Different types of heavyand light chains are recognized. The light chains are structurallydivided into two domains, a variable region (VL) and a constant region(CL). The heavy chain is also divided into distinct structural domains.For example, a γ heavy chain comprises, from the amino terminus, avariable region (VH), a constant region (CH1), a hinge region, a secondconstant region (CH2) and a third constant region (CH3).

The term “equivalent fusion polypeptide” refers to a fusion polypeptidewhich has an identical amino acid sequence compared to a referencefusion polypeptide, except for a KEX2 site pre-sequence. A first fusionpolypeptide having a first KEX2 site pre-sequence is equivalent to asecond fusion polypeptide having a different KEX2 site pre-sequence ifthe polypeptides have identical amino acid sequences, except for thedifference in the KEX2 site pre-sequence.

The term “derived” encompasses the terms “originated from”, “obtained”or “obtainable from”, and “isolated from”.

“Host strain” or “host cell” means a suitable host for an expressionvector or DNA construct comprising a polynucleotide encoding apolypeptide and particularly a recombinant fusion polypeptideencompassed by the invention. In specific embodiments, the host strainsmay be a filamentous fungal cell. The term “host cell” includes bothcells and protoplasts.

The term “filamentous fungi” refers to all filamentous forms of thesubdivision Eumycotina (See, Alexopoulos, C. J. (1962), INTRODUCTORYMYCOLOGY, Wiley, New York). These fungi are characterized by avegetative mycelium with a cell wall composed of chitin, glucans, andother complex polysaccharides. The filamentous fungi of the presentinvention are morphologically, physiologically, and genetically distinctfrom yeasts. Vegetative growth by filamentous fungi is by hyphalelongation and carbon catabolism is obligatory aerobic.

The term “culturing” refers to growing a population of microbial cellsunder suitable conditions in a liquid or solid medium.

The term “heterologous” with reference to a polynucleotide orpolypeptide refers to a polynucleotide or polypeptide that does notnaturally occur in a host cell. In some embodiments, the protein is acommercially important industrial protein and in some embodiments, theheterologous protein is a therapeutic protein. It is intended that theterm encompass proteins that are encoded by naturally occurring genes,mutated genes, and/or synthetic genes.

The term “homologous” with reference to a polynucleotide or proteinrefers to a polynucleotide or protein that occurs naturally in the hostcell.

The terms “recovered”, “isolated”, and “separated” as used herein referto a protein, cell, nucleic acid or amino acid that is removed from atleast one component with which it is naturally associated.

As used herein, the terms “transformed”, “stably transformed” and“transgenic” used in reference to a cell means the cell has a non-native(e.g., heterologous) nucleic acid sequence or additional copy of anative (e.g., homologous) nucleic acid sequence integrated into itsgenome or has an episomal plasmid that is maintained through multiplegenerations.

As used herein, the term “expression” refers to the process by which apolypeptide is produced based on the nucleic acid sequence of a gene.The process includes both transcription and translation.

The term “glycosylated” protein means a protein that has oligosaccharidemolecules added to particular amino acid residue on the protein.

The term “non-glycosylated” protein is a protein that does not haveoligosaccharide molecules attached to the protein.

The term “introduced” in the context of inserting a nucleic acidsequence into a cell, means “transfection”, or “transformation” or“transduction” and includes reference to the incorporation of a nucleicacid sequence into a eukaryotic or prokaryotic cell wherein the nucleicacid sequence may be incorporated into the genome of the cell (e.g.,chromosome, plasmid, plastid, or mitochondrial DNA), converted into anautonomous replicon, or transiently expressed (e.g., transfected mRNA).

The term “KEX2” refers to a calcium-dependent endopeptidase having anactivity defined as EC 3.4.21.61, according to IUBMB EnzymeNomenclature. KEX2 cleaves a peptide bond (the KEX2 cleavage site) thatis immediately C-terminal to a pair of basic amino acids during proteinsecretion.

The term “KEX2 region” refers to a contiguous eight to four amino acidresidue region which is located between the C-terminus end of a carrierprotein and the N-terminal end of a desired protein in a fusionpolypeptide. The KEX2 region is comprised of a KEX2 site and a KEX2 sitepre-sequence.

The term “KEX2 site” refers to a two amino acid KEX2 cleavage motif in aprotein. A KEX2 site contains two contiguous basic amino acids (e.g.,lysine, histidine and/or arginine) in any order, (e.g., KK, RR, KR orRK).

The term “KEX2 site pre-sequence” refers to the two to six contiguousamino acids [(X)_(n) where n is 2 to 6] immediately preceding (i.e.,immediately N-terminal to) the KEX2 site. For example, if a KEX2 regionis defined as VAVEKR, the “KR” motif is the KEX2 site of the region; nis 4 and the “VAVE” motif corresponds to the KEX2 site pre-sequence ofthe region.

The term “variant” refers to a region of a protein that contains one ormore different amino acids as compared to a reference protein.

The term “secreted protein” refers to a region of a polypeptide that isreleased from a cell during protein secretion. In some embodiments, thesecreted protein is the protein that is released or cleaved from arecombinant fusion polypeptide of the invention.

The term “secretion” refers to the selective movement of a proteinacross a membrane in a host cell to the extracellular space andsurrounding media.

The terms “determining”, “measuring”, “evaluating”, “assessing” and“assaying” are used interchangeably herein to refer to any form ofmeasurement, and include determining if an element is present or not.These terms include both quantitative and/or qualitative determinations.Assessing may be relative or absolute.

“Assessing the presence of” includes determining the amount of somethingpresent, as well as determining whether it is present or absent.

Other definitions of terms may appear throughout the specification.

DETAILED DESCRIPTION

Before the exemplary embodiments are described in more detail, it is tobe understood that this invention is not limited to particularembodiments described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, exemplary and preferred methods and materials are nowdescribed. All publications mentioned herein are incorporated herein byreference to disclose and describe the methods and/or materials inconnection with which the publications are cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “agene” includes a plurality of such candidate agents and reference to“the cell” includes reference to one or more cells and equivalentsthereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates, which may need to be independently confirmed.

Fusion Polypeptides—

As noted above, the subject fusion polypeptide comprises: a) a signalsequence; b) a carrier protein; c) a KEX2 region comprising: i) a KEX2site and ii) a KEX2 site pre-sequence immediately N-terminal to the KEX2site; and d) a desired protein.

FIG. 1 illustrates a subject fusion polypeptide of the invention. Thevarious parts of a subject polypeptide (i.e., “signal sequence”, carrierprotein, “KEX2 region” and “desired protein”) are so labeled solely forclarity and convenience. It is recognized that the subject fusionpolypeptide may also be referred to as a “pro-protein” or “precursorprotein” because it generally contains an N-terminal region that iscleaved off during secretion and a C-terminal region that is secreted.

Signal Sequences and Carrier Proteins—

The signal sequence of a subject fusion polypeptide may be any signalsequence that facilitates protein secretion from a filamentous fungalcell. In particular embodiments, the subject fusion polypeptide maycomprise a signal sequence for a protein that is known to be highlysecreted from the filamentous cell in which the fusion protein is to beproduced. The signal sequence employed may be endogenous ornon-endogenous to the cell in which the fusion polypeptide is to beproduced. In particular embodiments, the signal sequence may comprise a“carrier” that contains the signal sequence at its N-terminus, where thecarrier is at least an N-terminal portion of a protein that isendogenous to the cell and efficiently secreted by a cell.

Suitable signal sequences and carriers are known in the art (see, e.g.,Ward et al, Bio/Technology 1990 8:435-440; and Paloheimo et al, Appliedand Environmental Microbiology 2003 69: 7073-7082). Examples of suitablesignal sequences and carrier proteins include those of cellobiohydrolaseI, cellobiohydrolase II, endoglucanases I, II and III, α-amylase,aspartyl proteases, glucoamylase, phytase, mannanase, α and βglucosidases, bovine chymosin, human interferon and human tissueplasminogen activator and synthetic consensus eukaryotic signalsequences such as those described by Gwynne et al., (1987)Bio/Technology 5:713-719.

In some embodiments, if Trichoderma (e.g. T. reesei) is employed as ahost cell, the signal sequence or carrier of T. reesei mannanase I(Man5A, or MANI), T. reesei cellobiohydrolase II (Cel6A or CBHII),endoglucanase I (Cel7b or EGI), endoglucanase II (Cel5a or EGII),endoglucanase III (Cel12A or EGIII), xylanases I or II (XynIIa orXynIIb) or T. reesei cellobiohydrolase I (Cel7a or CBHI) may be employedin the fusion polypeptide.

In other embodiments, if an Aspergillus (e.g. A. niger) is employed as ahost cell, the signal sequence or carrier of A. niger glucoamylase(GlaA) or alpha amylase may be employed in the fusion polypeptide.Aspergillus niger and Aspergillus awamori glucoamylases have identicalamino acid sequences. Two forms of the enzyme are generally recognizedin culture supernatants. GAI is the full length form (amino acidresidues 1-616) and GAII is a natural proteolytic fragment comprisingamino acid residues 1-512. GAI is known to fold as two separate domainsjoined by an extended linker region. The two domains are the 471 residuecatalytic domain (amino acids 1-471) and the 108 residue starch bindingdomain (amino acids 509-616), the linker region between the two domainsbeing 36 residues (amino acids 472-508). GAII lacks the starch bindingdomain. Reference is made to Libby et al., (1994) Protein Engineering7:1109-1114. In some embodiments, the glucoamylase which is used as acarrier protein and including a signal sequence will have greater than95%, 96%, 97%, 98% and 99% sequence identity with a catalytic domain ofan Aspergillus or Trichoderma glucoamylase. The term “catalytic domain”refers to a structural portion or region of the amino acid sequence of aprotein which posses the catalytic activity of the protein.

In certain embodiments, the signal sequence and the carrier protein areobtained from the same gene. In some embodiments, the signal sequenceand the carrier protein are obtained from different genes.

The carrier protein may include all or part of the mature sequence of asecreted polypeptide. In some embodiments, full length secretedpolypeptides are used. However, functional portions of secretedpolypeptides may be employed. As used herein “portion” of a secretedpolypeptide or grammatical equivalents means a truncated secretedpolypeptide that retains its ability to fold into a normal, albeittruncated, configuration.

In some cases, the truncation of the secreted polypeptide means that thefunctional protein retains a biological function. In some embodiments,the catalytic domain of the secreted polypeptide is used, although otherfunctional domains could be used, for example the substrate bindingdomain. In one embodiment, when glucoamylase is used as the carrierprotein (i.e. glucoamylase from Aspergillus niger), preferred functionalportions retain the catalytic domain of the enzyme and include aminoacids 1-471 (see, WO 03089614, e.g., example 10). In another embodiment,when CBH I is used as the carrier protein (i.e. CBH I from Trichodermareesei) preferred functional portions retain the catalytic domain of theenzyme. Reference is made to SEQ ID NO:1 of FIG. 2 of WO 05093073,wherein the sequence encoding a Trichoderma reesei CBH1 signal sequence,T. reesei CBH1 catalytic domain (also referred to as catalytic core orcore domain) and T. reesei CBH1 linker is disclosed. In someembodiments, a CBH1 carrier protein and including a signal sequence willhave greater than 95%, 96%, 97%, 98% and 99% sequence identity with SEQID NO:1 of FIG. 2 of WO 05093073.

In general, if the carrier protein is a truncated protein, it isC-terminally truncated (i.e., contains an intact N-terminus).Alternatively, the carrier protein may be N-terminally truncated, oroptionally truncated at both ends to leave a functional portion.Generally such portions of a secreted protein which comprise a carrierprotein comprise greater than 50%, greater than 70%, greater than 80%and greater than 90% of the secreted protein and preferably theN-terminal portion of the secreted protein. In some embodiments, thecarrier protein will include a linker region in addition to thecatalytic domain. In the fusion constructs of the examples herein, partof the linker region of the CBHI protein was used in the carrierprotein.

As used herein, the first amino acid sequence comprising a signalsequence functional as a secretory sequence is encoded by a first DNAmolecule. The second amino acid sequence comprising the carrier proteinis encoded by a second DNA sequence. However, as described above thesignal sequence and the carrier protein may be obtained from the samegene.

KEX2 Region—

The KEX2 region comprises a KEX2 site (B₁B₂) and a KEX2 sitepre-sequence ((X)_(n=2-6)) immediately N-terminal to said KEX2 site. Insome embodiments the KEX2 region provides means for cleavage(separation) at the amino terminus of the desired protein from thefusion polypeptide in vivo. The KEX2 region of a fusion polypeptide ofthe encompassed by the invention is not a naturally occurring regionbetween the carrier protein and the desired protein.

The KEX2 cleavage site, which occurs at the C-terminal end of the KEX2region, may be cleaved by a native filamentous fungal protease (e.g. anative Aspergillus KEXB-like protease or native Trichoderma KEX2protease). The desired protein is cleaved from a fusion polypeptideaccording to the invention immediately downstream of the KEX2 site.

The KEX2 site contains amino acid sequence “B₁B₂” wherein B₁ and B₂, areindependently, basic amino acids. Preferably the KEX2 site includes anyone of KK, KR, RK or RR and more preferably is KR.

The KEX2 site pre-sequence comprises amino acid sequence (X)_(n=2-6)wherein X is any amino acid and n is 2 to 6 and preferably 4. The KEX2region as defined herein is not found naturally in the carrier proteinat the C-terminus of the carrier protein, which comprises the fusionpolypeptide according to the invention. In some embodiments, the KEX2site pre-sequence is an amino acid sequence that is different from thenaturally occurring contiguous (X)_(n=2-6) amino acid residues on theC-terminus of the carrier protein. However, the contiguous (X)₌₂₋₆ aminoacid residues may be found in other parts of the carrier protein and maybe linked with a KEX2 site (B₁B₂) but the KEX2 region will not beattached to the N-terminus of the desired protein.

In some embodiments, when the KEX2 site pre-sequence is defined asX₄X₃X₂X₁B₁B₂,

a) X₁, X₂ and X₃ are not G;

b) X₁ is not S, if X₂ and X₃ are G, X₄ is A, or X₃ is 5;

C) X₄ is not T, if X₃ is A and X₂ is S; or

d) X₁ is not D.

In some preferred embodiments, the KEX2 region is X₄X₃X₂X₁B₁B₂ whereinB₁B₂ is KR and

a) X₁, X₂ and X₃ are not G;

a) X₁ is not S, if X₂ and X₃ are G, X₄ is A, or X₃ is 5;

b) X₄ is not T, if X₃ is A and X₂ is S; or

c) d) X₁ is not D.

In other embodiments, the KEX2 site pre-sequence is defined as X₄X₃X₂X₁wherein,

a) X₄ is V, S, N, L, or K;

a) X₃ is A, V, D, W, E or P;

b) X₂ is V, I, L or F; and

c) X₁ is E, S, T or Y.

In yet other embodiments the KEX2 site pre-sequence is defined asX₄X₃X₂X₁ wherein,

a) X₄ is V, N, or L;

a) X₃ is A, V, D, W, E or P;

b) X₂ is V, I, L or F; and

c) X₁ is E or Y.

In yet further embodiments the X₄X₃X₂X₁KR KEX2 region may be selectedfrom the group of X₄ is V; X₃ is A; X₂ is V; X₁ is E or Y andcombinations thereof.

In some embodiments, the KEX2 site pre-sequence is selected from thegroup consisting of VAVE (SEQ ID NO: 84); NVIS (SEQ ID NO: 85); SDVT(SEQ ID NO: 86); VAVY (SEQ ID NO: 87); LAVE (SEQ ID NO: 88); KAVE (SEQID NO: 89); VAIE (SEQ ID NO: 90); VALE (SEQ ID NO: 91); VAFE (SEQ ID NO:92); VWVE (SEQ ID NO: 93); VEVE (SEQ ID NO: 94); and VPVE (SEQ ID NO:95).

In some embodiments, the KEX2 site pre-sequence is not KSRS (SEQ ID NO:109); SRIS (SEQ ID NO: 111); GGGS (SEQ ID NO: 110); TSTY (SEQ ID NO:96); ASIS (SEQ ID NO: 97); ATAS (SEQ ID NO: 98); TASQ (SEQ ID NO: 99);TASL (SEQ ID NO: 100), SVIS (SEQ ID NO: 101); NVIS (SEQ ID NO: 85); GGG;TSRD (SEQ ID NO: 102); SPMD (SEQ ID NO: 106); DLGE (SEQ ID NO: 107); orTPTA (SEQ ID NO: 108).

While the preferred KEX2 region is defined as X₄X₃X₂X₁B₁B₂, as indicatedabove, the KEX2 site pre-sequence can include 6 amino acid residues, insome embodiments, the KEX2 region may include one or two more amino acidresidues In other embodiments, the KEX2 site pre-sequence may includeonly 2 or 3 amino acid residues (X₃X₂X₁B₁B₂ or X₂X₁B₁B₂). In thisembodiment,

a) X₁, X₂ and X₃ are not G (e.g. GGGB₁B₂ or GGB₁B₂),

a) X₁ is not S, if X₂ and X₃ are G or X₃ is S (e.g. SX₂S or SGS); and

-   -   b) X₁ is not D.

In some embodiments, the KEX2 site pre-sequence provides for enhancedcleavage and/or secretion of a desired protein from a host cell ascompared to the cleavage and/or secretion of the desired protein from anequivalent fusion polypeptide lacking a KEX2 site pre-sequence.

In some embodiments, the KEX2 site pre-sequence is an optimized KEX2site pre-sequence. An optimized KEX2 pre-sequence is a KEX2 pre-sequenceencompassed by the invention but which provides greater or moreefficient cleavage or secretion from a host cell as compared to othervariant KEX2 site pre-sequences.

In some embodiments, the fusion polypeptide encompassed by the inventionwill include an optimized KEX2 pre-sequence as the KEX2 pre-sequence.The optimized KEX2 pre-sequence may be employed with any signalsequence, any carrier region from a secreted protein, any KEX2 site, orany desired protein. A subject KEX2 region containing an optimized KEX2site pre-sequence may be non-naturally occurring. In certainembodiments, a subject KEX2 region containing an optimized KEX2 sitepre-sequence is not found in any protein that is secreted from afilamentous fungal cell.

Desired Proteins—

The desired protein (or the carrier protein) may be any portion of aprotein that can be secreted from a filamentous fungal cell, whichproteins include, so called industrial enzymes, therapeutic proteins,hormones, structural proteins, plasma proteins, food additives andfoodstuffs and the like. The desired protein may be a heterologous orhomologous protein and may include hybrid polypeptides that comprise acombination of partial or complete polypeptides each of which may behomologous or heterologous with regard to the fungal expression host.The desired secreted protein may be derived from bacterial (e.g.Bacillus species and Pseudomonas species) fungal (e.g. Aspergillus,Trichoderma, Humicola, or Mucor species), viral (e.g. Hepatitis A or Bor Adenovirus), mammalian (e.g. human or mouse), and plant sources.Desired proteins include naturally occurring allelic variations ofproteins as well as engineered variations.

In one embodiment, the desired protein may be an enzyme such as acarbohydrase, such as a starch hydrolyzing α-amylase, an alkalineα-amylase, a β-amylase, a cellulase; a dextranase, an α-glucosidase, anα-galactosidase, a glucoamylase, a hemicellulase, a pentosanase, axylanase, an invertase, a lactase, a naringanase, a pectinase or apullulanase; a protease such as an acid protease, an alkali protease,bromelain, ficin, a neutral protease, papain, pepsin, a peptidase,rennet, rennin, chymosin, subtilisin, thermolysin, an asparticproteinase, or trypsin; a granular starch hydrolyzing enzyme, such as aglucoamylase or an alpha amylase; a lipase or esterase, such as atriglyceridase, a phospholipase, a pregastric esterase, a phosphatase, aphytase, an amidase, an iminoacylase, a glutaminase, a lysozyme, or apenicillin acylase; an isomerase such as glucose isomerase; a phenoloxidizing enzyme, e.g., a laccase; an oxidoreductases, e.g., an aminoacid oxidase, a catalase, a chloroperoxidase, a glucose oxidase, ahydroxysteroid dehydrogenase or a peroxidase; a lyase such as aacetolactate decarboxylase, a aspartic β-decarboxylase, a fumarese or ahistadase; a transferase such as cyclodextrin glycosyltranferase or anacyl transferase; or a ligase, for example. In particular embodiments,the protein may be an aminopeptidase, a carboxypeptidase, a chitinase, aglucoamylase, an alpha amylase, a cutinase, a phytase, adeoxyribonuclease, an α-galactosidase, a β-galactosidase, aβ-glucosidase, a laccase, a mannosidase, a mutanase, a pectinolyticenzyme, a polyphenoloxidase, ribonuclease or transglutaminase.

In other embodiments, the desired protein may be a therapeutic protein(i.e., a protein having a therapeutic biological activity). Examples ofsuitable therapeutic proteins include: erythropoietin, cytokines such asinterferon-α, interferon-β, interferon-γ, interferon-o, andgranulocyte-CSF, GM-CSF, coagulation factors such as factor VIII, factorIX, and human protein C, antithrombin III, thrombin, soluble IgEreceptor α-chain, immunoglobulin, such as immunoglobulin G (IgG), IgGfragments, IgG fusions, IgM or IgA; interleukins, urokinase, chymase,and urea trypsin inhibitor, IGF-binding protein, epidermal growthfactor, growth hormone-releasing factor, annexin V fusion protein,angiostatin, vascular endothelial growth factor-2, myeloid progenitorinhibitory factor-1, osteoprotegerin, α-1-antitrypsin, α-feto proteins,DNase II, kringle 3 of human plasminogen, glucocerebrosidase, TNFbinding protein 1, follicle stimulating hormone, cytotoxic T lymphocyteassociated antigen 4-Ig, transmembrane activator and calcium modulatorand cyclophilin ligand, soluble TNF receptor Fc fusion, glucagon likeprotein 1 and IL-2 receptor agonist.

In some preferred embodiments, the desired protein is an immunoglobulinfrom any class, G, A, M, E or D. (See, U.S. Pat. No. 4,816,567 andreferences cited therein for a discussion of immunoglobulin structure).In other preferred embodiments, the antibody proteins such as monoclonalantibodies including heavy or light chains and fragments thereof. Infurther embodiments, humanized antibodies are of particular interest asa desired protein (e.g. trastuzumab (herceptin)). Some specific examplesof preferred monoclonal antibody fragments are truncated forms of theheavy chain to remove part of the constant region such as Fab fragmentsin which the heavy chain (Fd) lacks the hinge region and the CH2 and CH3domains; Fab′ fragments in which the heavy chain includes the hingeregion but lacks the CH2 and CH3 domains; and F(ab′)₂ fragments whichincludes the Fab portion connected by the hinge region. (Verma et al.,(1998) J. Immunological Methods 216:165-181 and Pennell and Eldin (1998)Res. Immunol. 149:599-603). Also of interest are single chain antibodies(ScFv) and single domain antibodies (e.g., camelid antibodies).

In some particularly preferred embodiments a fusion polypeptideaccording to the invention will comprise in operable linkage a signalsequence; a carrier protein; a KEX2 region and a desired protein asindicated below:

Fusion DNA Constructs and Vectors—

In some embodiments, the invention provides a fusion DNA constructencoding a fusion polypeptide as disclosed above, comprising in operablelinkage from the 5′ end of said construct, a promoter; a first DNAmolecule encoding a signal sequence; a second DNA molecule encoding acarrier protein; a third DNA molecule encoding a KEX2 region, said KEX2region comprising a KEX2 site and a KEX2 site pre-sequence immediately5′ to the KEX2 site; and a fourth DNA molecule encoding a desiredprotein. Since the genetic code is known, the design and production ofthese nucleic acids is well within the skill of an artisan, given thedescription of the subject fusion polypeptide. In certain embodiments,the nucleic acids may be codon optimized for expression of the fusionpolypeptide in a particular host cell. Since codon usage tables areavailable for many species of filamentous fungi, the design andproduction of codon-optimized nucleic acids that encodes a subjectfusion polypeptide would be well within the skill of one of skill in theart.

Promoters—

Examples of suitable promoters for directing the transcription of asubject nucleic acid in a filamentous fungal host cell are promotersobtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucormiehei aspartic proteinase, Aspergillus niger neutral alpha-amylase,Aspergillus niger acid stable alpha-amylase (Korman et al (1990) Curr.Genet. 17:203-212; Crines et al., (1989) Gene 79: 107-117), Aspergillusniger or Aspergillus awamori glucoamylase (glaA) (Nunberg et al., (1984)Mol. Cell. Biol. 4:2306-2315; Boel E. et al., (1984) EMBO J. 3:1581-1585), Rhizomucor miehei lipase, Aspergillus oryzae alkalineprotease, Aspergillus oryzae triose phosphate isomerase, Aspergillusnidulans acetamidase (Hyner et al., (1983) Mol. Cell. Biol.3:1430-1439), Fusarium venenatum amyloglucosidase, Fusarium oxysporumtrypsin-like protease (WO 96/00787), Trichoderma reeseicellobiohydrolase I (Shoemaker et al. (1984) EPA EPO 0137280),Trichoderma reesei cellobiohydrolase II, Trichoderma reeseiendoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reeseiendoglucanase III, Trichoderma reesei endoglucanase IV, Trichodermareesei endoglucanase V, Trichoderma reesei xylanase I, Trichodermareesei xylanase II, Trichoderma reesei beta-xylosidase, as well as theNA2-tpi promoter (a hybrid of the promoters from the genes forAspergillus niger neutral alpha-amylase and Aspergillus oryzae triosephosphate isomerase); and mutant, truncated, and hybrid promotersthereof. Reference is also made to Yelton et al., (1984) Proc. Natl.Acad. Sci. USA 81:1470-1474; Mullaney et al., (1985) Mol. Gen. Genet.199:37-45; Lockington et al., (1986) Gene 33: 137-149; Macknight et al.,(1986) Cell 46: 143-147; Hynes et al., (1983) Mol. Cell. Biol. 3:1430-1439. Higher eukaryotic promoters such as SV40 early promoter(Barclay et al (1983) Molecular and Cellular Biology 3:2117-2130) mayalso be useful. Promoters may be constitutive or inducible promoters.Some preferred promoters include a Trichoderma reesei cellobiohydrolaseI or II, a Trichoderma reesei endoglucanase I, II or III, and aTrichoderma reesei xylanase II.

Vectors—

A subject polynucleotide may be present in a vector, for example, aphage, plasmid, viral, or retroviral vector. In certain embodiments, thevector may be an expression vector for expressing a subject fusionpolypeptide in a filamentous fungal cell.

Vectors for expression of recombinant proteins are well known in the art(Ausubel, et al, Short Protocols in Molecular Biology, 3rd ed., Wiley &Sons, 1995; Sambrook, et al., Molecular Cloning: A Laboratory Manual,Second Edition, (1989) Cold Spring Harbor, N.Y.).

A fusion DNA construct according to the invention may be constructedusing well known techniques as is generally described for example in EPOpublication 0 215 594.

Natural or synthetic polynucleotide fragments encoding for the desiredprotein (e.g. an immunoglobulin) may be incorporated into heterologousnucleic acid constructs or vectors, capable of introduction into andreplication in a filamentous fungal cell.

Once a DNA construct or more specifically a fusion DNA constructencompassed by the invention is made it may be incorporated into anynumber of vectors as is known in the art. While the DNA construct willpreferably include a promoter sequence, in some embodiments the vectorwill include other regulatory sequences functional in the host to betransformed, such as ribosomal binding sites, transcription start andstop sequences, terminator sequences, polyadenylation signals, enhancersand or activators. In some embodiments, a polynucleotide encoding thedesired protein and KEX2 region will be inserted into a vector whichcomprises a promoter, signal sequence and carrier protein at anappropriate restriction endonuclease site by standard procedures. Suchprocedures and related sub-cloning procedures are deemed to be withinthe scope of knowledge of those skilled in the art.

Terminator sequences which are recognized by the expression host toterminate transcription may be operably linked to the 3′ end of thefusion DNA construct encoding the fusion protein to be expressed. Thoseof general skill in the art are well aware of various terminatorsequences that may be used with filamentous fungi. Non-limiting examplesinclude the terminator from the Aspergillus nidulans trpC gene (YeltonM. et al., (1984) Proc. Natl. Acad. Sci. USA 81: 1470-1474) or theterminator from the Aspergillus niger glucoamylase genes (Nunberg et al.(1984) Mol. Cell. Biol. 4: 2306-2353) or the terminator from theTrichoderma reesei cellobiohydrolase I gene.

Polyadenylation sequences are DNA sequences which when transcribed arerecognized by the expression host to add polyadenosine residues totranscribed mRNA. Examples include polyadenylation sequences from A.nidulans trpC gene (Yelton et al (1984) Proc. Natl. Acad. Sci. USA81;1470-1474); from A. niger glucoamylase gene (Nunberg et al. (1984)Mol. Cell. Biol. 4:2306-2315); the A. oryzae or A. niger alpha amylasegene and the Rhizomucor miehei carboxyl protease gene. Any fungalpolyadenylation sequence is likely to be functional in the presentinvention.

In further embodiments, the fusion DNA construct or the vectorcomprising the fusion DNA construct will contain a selectable markergene to allow the selection of transformed host cells. Selection markergenes are well known in the art and will vary with the host cell used.Examples of selectable markers include but are not limited to ones thatconfer antimicrobial resistance (e.g. hygromycin, bleomycin,chloroamphenicol and phleomycin). Genes that confer metabolic advantage,such as nutritional selective markers also find use in the invention.Some of these markers include amdS. Also sequences encoding genes whichcomplement an auxotrophic defect may be used as selection markers (e.g.pyr4 complementation of a pyr4 deficient A. nidulans, A. awamori orTrichoderma reesei and argB complementation of an argB deficientstrain). Reference is made to Kelley et al., (1985) EMBO J. 4: 475-479;Penttila et al., (1987) Gene 61:155-164 and Kinghorn et al (1992)Applied Molecular Genetics of Filamentous Fungi, Blackie Academic andProfessional, Chapman and Hall, London.

Host cells—

A host cell comprising a fusion DNA construct according to the inventionis also provided. In certain embodiments, the host cell may be afilamentous fungal host cell. In some embodiments, the cells may befilamentous fungal cells of a strain that has a history of use forproduction of proteins that have GRAS status, i.e., a GenerallyRecognized as Safe, by the FDA.

In particular embodiments, the subject fungal cell may be a cell of thefollowing species: Trichoderma, (e.g., Trichoderma reesei (previouslyclassified as T. longibrachiatum and currently also known as Hypocreajecorina), Trichoderma viride, Trichoderma koningii, and Trichodermaharzianum)); Penicillium sp.: Humicola sp. (e.g., Humicola insolens andHumicola grisea); Chrysosporium sp. (e.g., C. lucknowense); Gliocladiumsp.; Aspergillus sp. (e.g., Aspergillus oryzae, Aspergillus niger,Aspergillus nidulans, Aspergillus kawachi, Aspergillus aculeatus,Aspergillus japonicus, Aspergillus sojae, and Aspergillus awamori),Fusarium sp.; Mucor sp.; Neurospora sp.; Hypocrea sp.; or Emericella sp.(See also, Innis et al., (1985) Sci. 228:21-26), among others. In someembodiments, subject fungal cells may be strains of Aspergillus oryzae,ATCC 11490, Aspergillus niger which include ATCC 22342, ATCC 44733, ATCC14331, NRRL 3112, and strains derived therefrom. In some embodiments,subject fungal cells may be strains of Trichoderma which includefunctional equivalents of RL-P37 (Sheir-Neiss et al. (1984) Appl.Microbiol. Biotechnology 20:46-53). Useful Trichoderma host strainsinclude; NRRL 15709, ATCC 13631, ATCC 26921 (QM 9414) ATCC 32098, ATCC32086, and ATCC 56765 (RUTC-30).

In some embodiments, a host cell may be one wherein native genes havebeen deleted or inactivated. In some embodiments, preferred host cellshave inactivated protease genes (e.g. aspartyl protease) and referenceis made to Berka et al. (1990) Gene 86:153-162 and U.S. Pat. No.6,509,171. In some embodiments, preferred host cells have inactivatedcellulase genes (e.g. cbh1, cbh2 and egl1, and egl2) and reference ismade to the quad deleted strain of T. reesei disclosed in WO 05/001036.

The above described fusion DNA construct may be present in the nucleargenome of the host cell or may be present in a plasmid that replicatesin the host cell, for example.

Transformation

Introduction of a DNA construct or vector into a host cell includestechniques such as transformation; electroporation; nuclearmicroinjection; transduction; transfection, (e.g., lipofection mediatedand DEAE-Dextrin mediated transfection); incubation with calciumphosphate DNA precipitate; high velocity bombardment with DNA-coatedmicroprojectiles; and protoplast fusion. General transformationtechniques are known in the art (See, e.g., Ausubel et al., (1987),supra, chapter 9; and Sambrook (1989) supra, and Campbell et al., (1989)Curr. Genet. 16:53-56). Reference is also made to WO 05/001036; U.S.Pat. No. 6,022,725; U.S. Pat. No. 6,103,490; U.S. Pat. No. 6,268,328;[and published U.S. patent applications 20060041113, 20060040353,20060040353 and 20050208623], which publications are incorporated hereinby reference.

The expression of recombinantly introduced proteins in Trichoderma isdescribed in U.S. Pat. No. 6,022,725; U.S. Pat. No. 6,268,328; Harkki etal. (1991); Enzyme Microb. Technol. 13:227-233; Harkki et al., (1989)Bio Technol. 7:596-603; EP 244,234; EP 215,594; and Nevalainen et al.,“The Molecular Biology of Trichoderma and its Application to theExpression of Both Homologous and Heterologous Genes”, in MOLECULARINDUSTRIAL MYCOLOGY, Eds. Leong and Berka, Marcel Dekker Inc., NY (1992)pp. 129-148). Reference is also made to Cao et al., (2000) Protein Sci.9:991-1001; Yelton et al., (1984) Proc. Natl. Acad. Sci. 81:1470-1471;U.S. Pat. No. 6,590,078; and Berka, et al., (1991) in: Applications ofEnzyme Biotechnology, Eds. Kelly and Baldwin, Plenum Press, NY) fortransformation of Aspergillus strains.

In one embodiment, the preparation of Trichoderma sp. for transformationinvolves the preparation of protoplasts from fungal mycelia. (See,Penttila et al., (1987) Gene 61:155-164). In some embodiments, themycelia are obtained from germinated vegetative spores.

Generally, cells are cultured in a standard medium containingphysiological salts and nutrients (See, e.g., Pourquie, J. et al.,BIOCHEMISTRY AND GENETICS OF CELLULOSE DEGRADATION, eds. Aubert, J. P.et al., Academic Press, pp. 71-86, 1988 and Ilmen, M. et al., (1997)Appl. Environ. Microbiol. 63:1298-1306). Common commercially preparedmedia (e.g., Yeast Malt Extract (YM) broth, Luria Bertani (LB) broth andSabouraud Dextrose (SD) broth also find use in the present invention.Preferred culture conditions for a given filamentous fungus are known inthe art and may be found in the scientific literature and/or from thesource of the fungi such as the American Type Culture Collection (ATCC)and Fungal Genetics Stock Center.

In some embodiments, when an immunoglobulin is the desired proteinimmunoglobulin expressing cells will be cultured under conditionstypically employed to culture the parental cell line. Generally, cellswill be cultured in standard medium containing physiological salts andnutrients such as that described by Ilmen et al., (1997) supra,. Cultureconditions will also be standard (e.g. incubation at 25-30° C. in shakeflasks on a rotary shaker) until desired levels of immunoglobulinexpression is achieved.

Protein Production Methods

Methods of producing a desired protein in a filamentous fungal cell arealso encompassed by the invention. In some embodiments these methodsinclude, obtaining a filamentous host cell comprising a fusion DNAconstruct or vector according to the invention and culturing thefilamentous host cell under suitable conditions which allow theexpression and secretion of the desired protein. While a culture of hostcells (i.e., a composition containing subject host cells and growthmedia) may contain the secreted protein of the fusion polypeptidedescribed above, in some embodiments the desired protein is recoveredfrom the culture media. In other embodiments, the desired protein ispurified. Protein may be recovered from growth media by any convenientmethod.

In some embodiments, a subject fungal cell may be cultured under batchor continuous fermentation conditions. A classical batch fermentation isa closed system, wherein the composition of the medium is set at thebeginning of the fermentation and is not subject to artificialalterations during the fermentation. Thus, at the beginning of thefermentation the medium is inoculated with the desired organism(s). Inthis method, fermentation is permitted to occur without the addition ofany components to the system. Typically, a batch fermentation qualifiesas a “batch” with respect to the addition of the carbon source andattempts are often made at controlling factors such as pH and oxygenconcentration. The metabolite and biomass compositions of the batchsystem change constantly up to the time the fermentation is stopped.Within batch cultures, cells progress through a static lag phase to ahigh growth log phase and finally to a stationary phase where growthrate is diminished or halted. If untreated, cells in the stationaryphase eventually die. In general, cells in log phase are responsible forthe bulk of production of end product.

A variation on the standard batch system is the “fed-batch fermentation”system, which also finds use with the present invention. In thisvariation of a typical batch system, the substrate is added inincrements as the fermentation progresses. Fed-batch systems are usefulwhen catabolite repression is apt to inhibit the metabolism of the cellsand where it is desirable to have limited amounts of substrate in themedium. Measurement of the actual substrate concentration in fed-batchsystems is difficult and is therefore estimated on the basis of thechanges of measurable factors such as pH, dissolved oxygen and thepartial pressure of waste gases such as CO₂. Batch and fed-batchfermentations are common and known in the art.

Continuous fermentation is an open system where a defined fermentationmedium is added continuously to a bioreactor and an equal amount ofconditioned medium is removed simultaneously for processing. Continuousfermentation generally maintains the cultures at a constant high densitywhere cells are primarily in log phase growth.

Continuous fermentation allows for the modulation of one factor or anynumber of factors that affect cell growth and/or end productconcentration. For example, in one embodiment, a limiting nutrient suchas the carbon source or nitrogen source is maintained at a fixed rateand all other parameters are allowed to moderate. In other systems, anumber of factors affecting growth can be altered continuously while thecell concentration, measured by media turbidity, is kept constant.Continuous systems strive to maintain steady state growth conditions.Thus, cell loss due to medium being drawn off must be balanced againstthe cell growth rate in the fermentation. Methods of modulatingnutrients and growth factors for continuous fermentation processes aswell as techniques for maximizing the rate of product formation areknown.

Expression and Secretion—

The production of a desired protein in a filamentous fungal cellcomprising a fusion DNA construct encoding a fusion polypeptide resultsin the secretion of the desired protein of the fusion polypeptide.During the secretion process in fungi, sugar chains may be attached to aprotein to be secreted to produce a glycosylated protein. In the presentinvention, the production of the desired protein, (e.g. an antibody),may include glycosylated or non-glycosylated protein.

In some embodiments, the secreted protein of the subject fusionpolypeptide is generally present in the culture medium of thefilamentous fungal cell at an amount that is higher than the amount ofthe desired secreted protein of an equivalent fusion polypeptide thatlacks the KEX2 site pre-sequence, produced by an equivalent filamentousfungal cell (i.e., the same cell type, grown under the same conditions).A culture of the subject cells producing a desired protein from a fusionpolypeptide according to the invention may contain more than 5%, morethan 10%, more than 20%, more than 40%, more than 60%, more than 80%,more than 100%, more than 150%, more than 200%, more than 300%, morethan 500%, and more than 1000% desired protein in the growth medium, ascompared to an equivalent cell culture that expresses an otherwiseequivalent protein that does not have a KEX2 site pre-sequence asencompassed by the invention.

In some embodiments, the level of expression and secretion for a desiredprotein (e.g. a full-length antibody) will be greater than 0.5 g/L.Routinely greater than 1.0 g/L of the desired protein may be recoveredfrom a culture media. Reproducible levels of greater than 1.5, 2.0 and3.0 g/L may be attained. In some embodiments, the level of expressionand secretion of the desired protein will be greater than 10 g/L andeven greater than 20 g/L.

In some embodiments of the invention, the cleavage of the desiredprotein from the recombinant fusion polypeptide will be greater than thecleavage of the same desired protein from an equivalent recombinantfusion polypeptide which lacks the KEX2 site pre-sequence. In someembodiments, the KEX2 site pre-sequence may result in a fusion proteinthat is cleaved to at least 80%, at least 85%, at least 90%, at least95%, at least 97%, at least 98%, at least 99% or 100% efficiency,wherein 100% efficiency results in a completely cleaved desiredsecretion protein from the fusion polypeptide.

In certain embodiments, the efficiency of protein cleavage may becalculated by determining amount of cleavage that has occurred, e.g., bydetermining the amount of cleaved versus the amount of uncleavedprotein. In one embodiment, the amount of protein cleavage may becalculated by determining the ratio of the amount of cleaved protein inthe growth medium to the amount of non-cleaved fusion protein in thegrowth medium per volume of cell culture.

A fusion polypeptide containing a KEX2 site pre-sequence or an optimizedKEX2 site pre-sequence may, in certain embodiments, result in a fusionpolypeptide that is cleaved to at least 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, 97%, 98%, 99% or 100% efficiency, wherein 100% efficiency is acompletely cleaved desired protein.

In other embodiments, the efficiency of secretion of a subject fusionpolypeptide may be calculated by determining the amount of the secretedportion of that fusion polypeptide in the growth medium of a cellsecreting that protein. This determination may be quantitative,qualitative, relative or absolute. In one embodiment, the amount ofsecreted protein in the growth medium of a cell secreting a subjectfusion may be at least 10%, at least 30%, at least 50%, at least 70%, atleast 90%, at least twice, at least five times, or at least ten timesgreater than the amount of the secreted protein secreted by a cellproducing an equivalent fusion polypeptide that does not contain anoptimized KEX2 pre-sequence.

In some embodiments the increase in secretion and/or cleavage may bemeasured against a standard KEX2 region defined as GGGB₁B₂, wherein B₁B₂is KK, KR, RK or RR and preferably KR. In an embodiment, the amount ofsecreted protein or desired protein in the growth medium of a cellsecreting a subject fusion may be at least 10%, at least 30%, at least50%, at least 70%, at least 80%, at least 90%, at least 100%, at least2×, at least 3×, at least 5×, and at least 10× greater than the amountof the secreted protein or desired protein secreted by an equivalentfusion polypeptide in an equivalent host under essentially the sameconditions.

Screening Methods—

Screening methods for identifying optimized KEX2 site pre-sequences arealso provided. These methods may include: a) altering a KEX2 sitepre-sequence of a parental fusion polypeptide to produce a testpolypeptide and b) evaluating secretion of the test fusion polypeptideby a filamentous fungal cell. In certain embodiments, the secretionand/or cleavage of the desired protein from test fusion polypeptides iscompared to the secretion and/or cleavage of the parental fusionprotein. In particular embodiments, the method includes evaluating theamount of a secreted protein of the fusion polypeptide in a growthmedium relative to the amount of a secreted portion of the fusionpolypeptide in a cell, per volume of culture, or assessing the amount ofa secreted protein of a recombinant fusion polypeptide in a growthmedium. In another embodiments the method includes evaluating the amountof a secreted protein (desired protein) released or cleaved from afusion polypeptide in a growth medium relative to the amount of thesecreted protein that remains in the form of the fusion polypetide (e.g.attached to the carrier protein).

In these screening assays, the parental fusion protein has an amino acidsequence that is schematically illustrated in FIG. 1, where X is anyamino acid. In certain embodiments, the parental fusion protein and thetest fusion protein may be identical except for their KEX2 sitepre-sequences. A parental recombinant fusion protein and a testrecombinant fusion protein may differ in one, two, three or four aminoacids in the KEX-2 site pre-sequence. An alteration may be an amino acidsubstitution, insertion or deletion, and if there are two or threealterations, the alterations may be in contiguous amino acids,non-contiguous amino acids, or a combination of contiguous andnon-contiguous amino acids.

In one embodiment, the KEX2 site pre-sequence of a parental fusionpolypeptide may be altered to produce a plurality of different testfusion polypeptides that each contains different KEX2 sitepre-sequences, and then evaluating secretion and/or cleavage of the testfusion polypeptides and the parental fusion polypeptides by afilamentous fungal cell.

These methods may be performed using protocols that are generally known(see, e.g., Ward et al (1990) Bio/Technology 8:435-440 and Spencer(1998) Eur. J. Biochem 258: 107-112, among others), in which a vector isintroduced into a cell, the cell is cultured, and the cell culture isassayed for the presence of the cellular protein. In one embodiment, arecombinant nucleic acid encoding a parent fusion (the structure ofwhich is shown in FIG. 1) is altered to produce a nucleic acid encodinga test polypeptide, and the two nucleic acids are used to transformidentical filamentous fugal cells (which may be any of the host cellslisted above). The two cell lines are cultured under identicalconditions, and the efficiency of secretion and/or cleavage of thesecreted portion of the protein is evaluated. The signal sequence,secreted protein, the KEX2 site and the KEX2 site pre-sequence of theparental fusion protein may be any known signal sequence, secretedprotein, KEX2 site or KEX2 site pre-sequence, including those listedabove.

As noted above, the efficiency of protein secretion or cleavage may beevaluated in many different ways, for example, by comparing the absoluteor normalized amounts of secreted portion in growth media between thedifferent cultures, or by comparing the amount of the secreted portionof the protein to the amount of the unsecreted portion of the protein.This evaluation may be quantitative, qualitative, relative or absolute,for example.

An optimized KEX2 site pre-sequence may be identified by testing aplurality of different test fusion proteins according to the abovemethods; and determining which of the different test fusion proteins issecreted and/or cleaved most efficiently; wherein the optimized KEX2site pre-sequence is the KEX2 site pre-sequence of the test recombinantfusion protein that is secreted most efficiently.

A culture of cells that contains at least 10%, at least 20%, at least30%, at least 50%, at least 70% and at least 95% more secreted or moredesired protein than a control culture indicates that KEX2 sitepre-sequence increases protein secretion and/or cleavage from thosecells.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade (°C.), pressure is at or near atmospheric and the following abbreviationsapply, M (Molar); μM (micromolar); N (Normal); mol (moles); mmol(millimoles); μmol (micromoles); nmol (nanomoles); g (grams); mg(milligrams) kg (kilograms); μg (micrograms); L (liters); ml(milliliters); h (hours); min (minutes); PAGE (polyacrylamide gelelectrophoresis); kDa (kilodaltons); and by (base pairs). The followingassays and methods are used in the examples provided below:

A. Construction of the pTrex4 Vector:

Synthetic DNA was cloned into a Trichoderma expression vector (pTrex4)to generate appropriate expression plasmids for use in the examplesdescribed below.

PTrex4 is a modified version of pTrex2 and derived from a pTrex3gexpression vector. The construction of pTrex3g is described in detail inExample 6 of WO 05/001036. In brief, the pTrex3g is based on the E. colivector pSL1180 (Pharmacia, Inc., Piscataway, N.J.) which is a pUC118plasmid based vector with an extended multiple cloning site containing64 hexamer restriction enzyme recognition sequences. It was designed asa Gateway destination vector (Hartley, J. L. et al., (2000) GenomeResearch 10:1788-1795) to allow insertion using Gateway Technology(Invitrogen) of any desired open reading frame between the promoter andterminator regions of the T. reesei cbh1 gene.

The details of pTrex4-her2 light chain DNA2.0 are as follows (FIG. 2 andFIG. 3): The plasmid is 10885 kb in size (SEQ ID NO: 103). Inserted intothe polylinker region of pSL1180 are the following segments of DNA:

A 2.2 bp segment of DNA from the promoter region of the T. reesei cbh1;DNA sequence of T. reesei cbh1 signal sequence (underlined); catalyticdomain; linker (italics) (1570 bases) (SEQ ID NO: 104)

ATGTATCGGAAGTTGGCCGTCATCTCGGCCTTCTTGGCCACAGCTCGTGCTCAGTCGGCCTGCACTCTCCAATCGGAGACTCACCCGCCTCTGACATGGCAGAAATGCTCGTCTGGTGGCACTTGCACTCAACAGACAGGCTCCGTGGTCATCGACGCCAACTGGCGCTGGACTCACGCTACGAACAGCAGCACGAACTGCTACGATGGCAACACTTGGAGCTCGACCCTATGTCCTGACAACGAGACCTGCGCGAAGAACTGCTGTCTGGACGGTGCCGCCTACGCGTCCACGTACGGAGTTACCACGAGCGGTAACAGCCTCTCCATTGGCTTTGTCACCCAGTCTGCGCAGAAGAACGTTGGCGCTCGCCTTTACCTTATGGCGAGCGACACGACCTACCAGGAATTCACCCTGCTTGGCAACGAGTTCTCTTTCGATGTTGATGTTTCGCAGCTGCCGTAAGTGACTTACCATGAACCCCTGACGTATCTTCTTGTGGGCTCCCAGCTGACTGGCCAATTTAAGGTGCGGCTTGAACGGAGCTCTCTACTTCGTGTCCATGGACGCGGATGGTGGCGTGAGCAAGTATCCCACCAACACCGCTGGCGCCAAGTACGGCACGGGGTACTGTGACAGCCAGTGTCCCCGCGATCTGAAGTTCATCAATGGCCAGGCCAACGTTGAGGGCTGGGAGCCGTCATCCAACAACGCAAACACGGGCATTGGAGGACACGGAAGCTGCTGCTCTGAGATGGATATCTGGGAGGCCAACTCCATCTCCGAGGCTCTTACCCCCCACCCTTGCACGACTGTCGGCCAGGAGATCTGCGAGGGTGATGGGTGCGGCGGAACTTACTCCGATAACAGATATGGCGGCACTTGCGATCCCGATGGCTGCGACTGGAACCCATACCGCCTGGGCAACACCAGCTTCTACGGCCCTGGCTCAAGCTTTACCCTCGATACCACCAAGAAATTGACCGTTGTCACCCAGTTCGAGACGTCGGGTGCCATCAACCGATACTATGTCCAGAATGGCGTCACTTTCCAGCAGCCCAACGCCGAGCTTGGTAGTTACTCTGGCAACGAGCTCAACGATGATTACTGCACAGCTGAGGAGGCAGAATTCGGCGGATCCTCTTTCTCAGACAAGGGCGGCCTGACTCAGTTCAAGAAGGCTACCTCTGGCGGCATGGTTCTGGTCATGAGTCTGTGGGATGATGTGAGTTTGATGGACAAACATGCGCGTTGACAAAGAGTCAAGCAGCTGACTGAGATGTTACAGTACTACGCCAACATGCTGTGGCTGGACTCCACCTACCCGACAAACGAGACCTCCTCCACACCCGGTGCCGTGCGCGGAAGCTGCTCCACCAGCTCCGGTGTCCCTGCTCAGGTCGAATCTCAGTCTCCCAACGCCAAGGTCACCTTCTCCAACATCAAGTTCGGACCCATTGGCAGCACCGGCAACCCTAGCGGCGGCAACCCTCCCGGCGGAAACCCGCCTGGCACCACCACCACCCGCCGCCCAGCCACTACCACTGGAAGCTCTCCCGGACCTACTAGT

The amino acid sequence of the T. reesei cbh1 signal sequence; catalyticdomain; linker (480 amino acids) is represented below (SEQ ID NO: 105)

MYRKLAVISAFLATARAQSACTLQSETHPPLTWQKCSSGGTCTQQTGSVVIDANWRWTHATNSSTNCYDGNTWSSTLCPDNETCAKNCCLDGAAYASTYGVTTSGNSLSIGFVTQSAQKNVGARLYLMASDTTYQEFTLLGNEFSFDVDVSQLPCGLNGALYFVSMDADGGVSKYPTNTAGAKYGTGYCDSQCPRDLKFINGQANVEGWEPSSNNANTGIGGHGSCCSEMDIWEANSISEALTPHPCTTVGQEICEGDGCGGTYSDNRYGGTCDPDGCDWNPYRLGNTSFYGPGSSFTLDTTKKLTVVTQFETSGAINRYYVQNGVTFQQPNAELGSYSGNELNDDYCTAEEAEFGGSSFSDKGGLTQFKKATSGGMVLVMSLWDDYYANMLWLDSTYPTNETSSTPGAVRGSCSTSSGVPAQVESQSPNAKVTFSNIKFGPIGSTGNPSGGNPPGGNPPGTTTTRRPATTTGSSPGPTS

The plasmid also contains a cbh1 terminator, an A. nidulans amdSselectable marker and nucleotides encoding the antibody light chain.

B. Biolistic Transformation of T. reesei:

In all examples below transformation was performed on a derivative ofthe quad deleted (Δchb1, Δcbh2, Δegl1, and Δegl2) T. reesei strain (WO05/001036) originally derived from RL-P37 (Sheir-Neiss et al., (1984)Appl. Microbiol. Biotechnol. 20:46-53; U.S. Pat. No. 4,797,361) with theappropriate pTrex4 vector using the protocol outlined below.

A suspension of spores (approximately 5×10⁸ spores/ml) from theTrichoderma strain was prepared. 100 ul-200 ul of spore suspension wasspread onto the center of plates of MM acetamide medium. MM acetamidemedium had the following composition: 0.6 g/L acetamide; 1.68 g/L CsCl;20 g/L glucose; 20 g/L KH₂PO₄; 0.6 g/L CaCl₂.2H₂O; 1 ml/L 1000× traceelements solution; 20 g/L Noble agar; pH 5.5. 1000× trace elementssolution contained 5.0 g/l FeSO₄.7H₂O, 1.6 g/l MnSO₄.H₂O, 1.4 g/lZnSO₄.7H₂O and 1.0 g/l CoCl₂.6H₂O. The spore suspension was allowed todry on the surface of the MM acetamide medium.

Transformation of the Trichoderma strain by the biolistic transformationmethod was accomplished using a Biolistic® PDS-1000/He Particle DeliverySystem from Bio-Rad (Hercules, Calif.) following the manufacturersinstructions (see, WO 05/001036 and US 2006/0003408).

C. Transformation of Aspergillus-

The Aspergillus transformation protocol was a modification of theCampbell method (Campbell et at. (1989). Curr. Genet. 16:53-56). Alsodetails of the transformation method for Aspergillus niger are disclosedin WO 03089614 and U.SPat. Pub. 20050153399. Transformants were assayedfor protein production on SDS gel and Western blot to select thetransformants based on the amount of protein produced.

D. Fermentation of T. reesei and Aspergillus niger Strains Transformedwith the Expression Vector:

In general the fermentation protocol as described in Foreman et al.,(2003) J. Biol. Chem. 278:31988-31997 was followed.

E. Proflo Media contains: 30 g/L α-lactose; 6.5 g/L (NH₄)₂SO₄; 2 g/LKH₂PO₄; 0.3 g/L MgSO₄.7H₂O; 0.2 g/L CaCl₂; 1 ml/L 1000× trace elementsalt solution; 2 ml/L 10% Tween 80; 22.25 g/L Proflo cottonseed flour(Traders Protein, Memphis, Tenn.); 0.72 g/L CaCO₃.F. Defined Media contains: 5 g/L (NH₄)₂SO₄; 33 g/L PIPPS buffer; 9 g/Lcasamino acids; 4.5 g/L KH₂PO₄; 1 g/L CaCl₂; 1 g/L MgSO₄.7H₂O; 5 ml/LMazu DF60-P antifoam (Mazur Chemicals, Gurnee, Ill.); 1 ml/L 1000× traceelements solution. After autoclaving 40 ml of 40% lactose was added.G. 1000× trace elements solution contains: 5.0 g/l FeSO₄.7H₂O, 1.6 g/lMnSO₄.H₂O, 1.4 g/l ZnSO₄.7H₂O and 1.0 g/l CoCl₂.6H₂OH. Protein Analysis was accomplished by standard SDS gel and Westernblot analysis.

Example 1 Construction of a Trastuzumab (Light Chain Expression StrainContaining a KRGGG (SEQ ID NO: 2) KEX2 cleavage site

DNA (SEQ ID NO:1) encoding the light chain of trastuzumab according tothe published amino acid sequence of antibody 4D5-8 (Carter et al, Proc.Natl. Acad. Sci. 1992 89: 4285-4289) was synthesized by DNA2.0 Inc.(1455 Adams Drive, Menlo Park, Calif. 94025).

(SEQ ID NO: 1) ACTAGTAAACGCGGTGGCGGTGATATTCAAATGACACAATCTCCTTCTTCTCTGTCAGCCTCAGTGGGCGACCGTGTGACGATTACTTGCCGCGCCTCTCAGGACGTTAACACTGCCGTCGCATGGTACCAGCAGAAGCCAGGCAAGGCGCCCAAGCTTCTGATTTACAGCGCTTCGTTCCTGTACTCTGGCGTGCCATCCCGCTTCTCTGGCAGCCGAAGCGGCACGGATTTCACCCTGACCATTTCGTCCCTGCAGCCCGAGGATTTCGCCACGTATTACTGCCAGCAGCACTACACCACTCCACCCACCTTTGGCCAAGGAACGAGAGTCGAAATCACTCGCACGGTCGCTGCCCCTTCAGTCTTCATCTTCCCCCCCAGCGACGAACAGCTGAAGTCTGGTACGGCCAGCGTCGTTTGCTTGCTTAATAACTTCTATCCGCGAGAGGCGAAGGTCCAATGGAAGGTTGATAACGTTCTGCAGTCCGGCAATTCGCAGGAGAGCGTGACCGAGCAGGATTCAAAGGATAGCACCTACTCACTCAGCAGCACCCTGACGTTGTCCAAGGCCGATTACGAGAAGCATAAGTTGTATGCATGCGAGGTCACCCACCAGGGACTGTCAAGCCCAGTTACCAAGTCGTTCAATCGAGGCGAGTGCTAAGGCGCGCC.

The light chain encoded by the DNA contains a KRGGG (SEQ ID NO:2) KEX2cleavage site at its N-terminal end. The restriction sites SpeI and AscIwere included for cloning proposes. The synthetic DNA was cloned intoTrichoderma expression vector (pTrex4) to generate an expression plasmidnamed pTrex4-her2 light chain DNA2.0 (FIG. 2). The resultant plasmidencodes a fusion protein containing a Trichoderma CBHI core/linkerregion and the antibody light chain, separated by a KEX2 site. Theplasmid was digested with XbaI restriction enzyme and transformedbiolistically into a Trichoderma reesei strain derived from the quaddeleted strain described in WO 05001036, example 5). More than 20transformants were obtained and transferred to new plates. Twenty stabletransformants were selected to grow in Proflo media for 2 days at 30° C.5 mls of 2 days old culture from Proflo were transferred to 50 mls ofDefine media. The cultures were grown for 5 days at 28° C. Culturebroths were centrifuged and supernatants were used for protein analysis.Western blot data (FIG. 4) indicated that more than 90% of the fusionprotein was cleaved in the best light chain producing strain(transformant1010-18, KRGGG variant). However, GGG will remain at theN-terminus of the cleaved antibody light chain which is undesirable. Aband of about 50 kd was also detected in Western blot, which may resultfrom dimerization of two light chain molecules.

Example 2 Construction of a Trastuzumab Light Chain Expression StrainContaining the GGGKR (SEQ ID NO: 5) KEX2 Cleavage Site

Two primers (GGACTAGTGGTGGCGGTAAACGCGATATTCAAATGACACAATCT C; SEQ ID NO:3and AAGGCGCGCCTTAGCACTCGCCTCGATTG; SEQ ID NO:4) were synthesized byInvitrogen (1600 Faraday Avenue. Carlsbad, Calif. 92008) and used toamplify trastuzumab light chain DNA.

The resulting PCR fragment encodes the antibody light chain containing aGGGKR (SEQ ID NO:5) sequence kex2 site at its N-terminal end. The PCRfragment was digested with restriction enzymes SpeI and AscI and clonedto expression Vector pTrex4 to generate a plasmid named aspTrex4-GGGKR-her2 DNA2.0. Fidelity of the PCR fragment was analyzed byDNA sequencing. The plasmid was digested with XbaI restriction enzymeand transformed biolistically using standard techniques into the T.reesei strain described above. More than 20 transformants were obtainedand transferred to new plates. A total of 21 stable transformants wereselected to grow in Proflo media for 2 days at 30° C. 5 mls of 2 daysold culture from Proflo were transferred to 50 mls of Define media. Thecultures were grown for 5 days at 28° C. Culture broths were centrifugedand supernatants were used. Western blot indicated that, more than 95%of the protein from transformant 1010-B5 (GGGKR variant) andtransformant 1010-B6 (GGGKR variant), was an uncleaved fusion protein(FIG. 4). A band of about 150 kd was also detected in Western blot. Itmay result from dimerization of two CBH1 core-light chain fusionmolecules.

Example 3 Construction of a Trastuzumab Light Chain Expression StrainContaining a GGGKRGGG (SEQ ID NO: 7) KEX2 Cleavage Site

Two oligos, GGACTAGTGGCGGTGGCAAACGCGGTGGCGGTGATATTC (SEQ ID NO. 6) andAAGGCGCGCCTTAGCACTCGCCTCGATTG (SEQ ID NO. 4), were synthesized byInvitrogen and used to amplify light chain DNA. The resulting PCRfragment encodes light chain and GGGKRGGG (SEQ ID NO:7) sequence forkex2 cleavage. The PCR fragment was digested with restriction enzymesSpeI and AscI and cloned to expression Vector pTrex4 to generate aplasmid named as pTrex4-GGGKRGGG-her2 light chain DNA2.0. Fidelity ofthe PCR fragment was analyzed by DNA sequencing. The plasmid wasdigested with XbaI restriction enzyme and transformed biolistically intothe T. reesei strain as described above. More than 10 transformants wereobtained and transferred to new plates. 3 stable transformants wereselected to grow in Proflo media for 2 days at 30° C. 5 mls of 2 daysold culture from Proflo were transferred to 50 mls of Define media. Thecultures were grown for 5 days at 28° C. Culture broths were centrifugedand supernatants were used for protein analysis. Western gel dataindicated that, in transformant 1011-1 (GGGKRGGG variant), more than 90%of the fusion protein was cleaved (FIG. 4). However, GGG remained at theN-terminus of the cleaved antibody light chain which is undesirable.

Example 4 Construction of a Trastuzumab Light Chain Expression StrainContaining a VAVEKR (SEQ ID NO: 9) KEX2 Region

A VAVEKR (SEQ ID NO: 9) KEX2 region is found naturally in the proregionof the T. reesei high pI xylanase, Xyn2 (Torronen et al., (1992)Biotechnol. 10:1461-1465). To construct a fusion polypetide according tothe invention, two oligos, GGACTAGTGTCGCCGTTGAGAAACGCGATATTCAAATGACACAATCTCC (SEQ ID NO. 8) and AAGGCGCGCCTTAGCACTCGCCTCGATTG (SEQ ID NO. 4),were synthesized by Invitrogen and used to amplify light chain DNA.

The resulting PCR fragment encodes light chain and VAVEKR (SEQ ID NO:9)sequence for kex2 cleavage. The PCR fragment was digested withrestriction enzymes SpeI and AscI and cloned to expression Vector pTrex4to generate a plasmid named as pTrex4-VAVE-her2 light chain DNA2.0.Fidelity of the PCR fragment was analyzed by DNA sequencing. The plasmidwas digested with XbaI restriction enzyme and transformed biolisticallyinto the T. reesei strain as described above. More than 20 transformantswere obtained and transferred to new plates. 6 stable transformants wereselected to grow in Proflo media for 2 days at 30° C. 5 mls of 2 daysold culture from Proflo were transferred to 50 mls of Define media. Thecultures were grown for 5 days at 28° C. Culture broths were centrifugedand supernatants were used for protein analysis. Western gel dataindicated that, in transformant1012-2 (VAVEKR variant, SEQ ID NO: 9),more than 95% of the fusion proteins were cleaved (FIG. 4).

Example 5 Construction of a Trastuzumab Light Chain Expression StrainContaining Variants of the VAVEKR (SEQ ID NO: 9) KEX2 Region

DNA (SEQ ID NO: 10) encoding the trastuzumab antibody light chain wassynthesized by Geneart (Josef-Engert-Strasse 11, 93053 Regesburg,Germany).

(SEQ ID NO: 10) ACTAGTAAGCGCGGCGGCGGCGAGGTCCAGCTCGTCGAGAGCGGCGGCGGCCTCGTCCAGCCCGGCGGCAGCCTCCGCCTCAGCTGCGCCGCCAGCGGCTTCAACATCAAGGACACCTACATCCACTGGGTCCGCCAGGCCCCCGGCAAGGGCCTCGAGTGGGTCGCCCGCATCTACCCCACCAACGGCTACACCCGCTACGCCGACAGCGTCAAGGGCCGCTTCACCATCAGCGCCGACACCAGCAAGAACACCGCCTACCTCCAGATGAACAGCCTCCGCGCCGAGGACACCGCCGTCTACTACTGCAGCCGCTGGGGCGGCGACGGCTTCTACGCCATGGACTACTGGGGCCAGGGCACCCTCGTCACGGTCTCCAGCGCCAGCACCAAGGGCCCAAGCGTCTTTCCCCTCGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACCGCCGCCCTCGGCTGCCTCGTCAAGGACTACTTCCCCGAGCCCGTCACTGTCAGCTGGAACAGCGGCGCTCTCACCAGCGGCGTCCACACCTTCCCCGCCGTCCTCCAGAGCAGCGGCCTCTACAGCCTCAGCAGCGTCGTCACCGTCCCCAGCAGCAGCCTCGGCACCCAGACCTACATCTGCAACGTCAACCACAAGCCCAGCAACACCAAGGTCGACAAGCGCGTCGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCCGCCCCCGAGCTGCTCGGCGGCCCCTCCGTCTTTCTCTTCCCCCCCAAGCCCAAGGACACCCTCATGATCAGCCGCACCCCCGAGGTCACCTGCGTCGTCGTCGATGTCAGCCACGAGGACCCCGAGGTCAAGTTCAACTGGTACGTCGACGGCGTCGAGGTCCACAACGCCAAGACCAAGCCCCGCGAGGAGCAGTACAACAGCACCTACCGCGTCGTCAGCGTCCTGACCGTCCTCCACCAGGACTGGCTCAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAGGCCCTCCCCGCCCCCATCGAA AAGACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAGCCCCAGGTCTACACCCTCCCCCCCAGCCGCGAGGAGATGACCAAGAACCAGGTCTCCCTCACCTGCCTGGTCAAGGGCTTCTACCCCAGCGACATCGCCGTCGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCGTCCTCGACAGCGACGGCAGCTTCTTCCTCTACAGCAAGCTCACCGTCGACAAGAGCCGCTGGCAGCAGGGCAACGTCTTTAGCTGCAGCGTCATGCACGAGGCCCTCCACAACCACTACACCCAGAAGAGCCTCAGCCTCAGCCCCGGCAAGTAAGGCGCG

This DNA encodes KRGGG (SEQ ID NO: 2) and the human antibody lightchain. Two restriction sites SpeI and AscI were included for cloningproposes. The nucleotide sequence was mutated to remove an internal kex2site by site-direct mutagenesis (Stratagene, 11011 North Torrey PinesRoad, La Jolla, Calif. 92037) and two primers used for the mutagenesisin the PCR reaction are TCGAGATCACCCGCACCGTCGCG

GCGCCAAG (SEQ ID NO: 11) and CGACGGTGCGGGTGATCTCGACCTTGGTGCCCTGG

CCG (SEQ ID NO: 12). The resulting light chain encoding DNA containedtwo substituted nucleotides at the DNA sequence which changed amino acidK to T.Two oligos, GGACTAGTGTCGCCGTTGAGAAACGCGACATCCAGATGACCCAGAGC (SEQ ID NO:13)and CTAAAGGGAACAAAAGCTGGAGC (SEQ ID NO: 14), were synthesized byInvitrogen and used to amplify light chain DNA. The resulting PCRfragment encodes light chain and VAVEKR (SEQ ID NO: 9). The PCR fragmentwas digested with restriction enzymes SpeI and AscI and cloned toexpression Vector pTrex4 to generate a plasmid named as pTrex4-VAVE-her2light chain Geneart (KR-TR). Fidelity of the PCR fragment was analyzedby DNA sequencing. The plasmid was digested with XbaI restriction enzymeand co-transformed biolistically into the T. reesei strain with heavychain expression plasmid. More than 40 transformants were obtained andtransferred to new plates. More than 20 stable transformants wereselected to grow in Proflo media for 2 days at 30° C. 5 mls of 2 daysold culture from Proflo were transferred to 50 mls of Define media. Thecultures were grown for 4 days at 28° C. Culture broths were centrifugedand supernatants were used for protein analysis. Western blot dataindicated that in the VAVEKR variant (transformant 17-43), more than 90%of the fusion protein was cleaved (FIG. 5).

To generate amino acid changes at the glutamine residue of the KEX2 sitepre-sequence of VAVEKR (SEQ ID NO: 9), a degenerate primer(GGACTAGTGTCGCCGTTNNSAAACGCGACATCC AGATGACCCAGAG (SEQ ID NO:15) wassynthesized and used in a PCR reaction with reverse primer (SEQ IDNO:14) to amplify DNA to generate a pool of PCR fragments. The mixed PCRfragments were cloned into Trichoderma expression vector (pTrex4). 13clones were sequenced and 7 variants were produced (table 1). All 7plasmids were transformed biolistically into the T. reesei strain. Morethan 40 transformants were obtained for each variant and transferred tonew plates. For the first set of three variants (VAVWKR (SEQ ID NO: 25),VAVGKR (SEQ ID NO: 26) and VAVRKR (SEQ ID NO: 27)), 15 stabletransformants for each variant were selected. For the second set of fourvariants (VAVTKR (SEQ ID NO: 28), VAVVKR (SEQ ID NO: 29), VAVAKR (SEQ IDNO: 30) and VAVLKR (SEQ ID NO: 31)), 11 stable transformants for eachvariant were selected. The selected transformants were grown in Proflomedia for 2 days at 28° C. 5 mls of 2 days old culture from Proflo weretransferred to 50 mls of Define media. The cultures were grown for 4days at 28° C. Culture broths were centrifuged and supernatants wereused for protein analysis.

A new primer (GGACTAGTGTCGCCGTTNACAAACGCGACATCCAGATGAC CCAGAG SEQ ID NO:16) was synthesized and used in a PCR reaction with reverse primer (SEQID NO: 14) to amplify DNA to generate PCR fragments with multiplesequences. The mixed PCR fragments were cloned into Trichodermaexpression vector (pTrex4). 10 clones were sequenced and 4 more variants(VAVDKR (SEQ ID NO: 32), VAVNKR (SEQ ID NO: 33), VAVYKR (SEQ ID NO: 34)and VAVHKR (SEQ ID NO: 35)) were produced. The plasmids were transformedbiolistically into the Trichoderma strain described above. More than 40transformants for each variant were obtained and transferred to newplates. 10 stable transformants for each variant were selected and grownin Proflo media for 2 days at 28° C. 5 mls of 2 days old culture fromProflo were transferred to 50 mls of Define media. The cultures weregrown for 4 days at 28° C. Culture broths were centrifuged andsupernatants were used for protein analysis.

One transformant (the best light chain producing transformant) from eachvariant at the glutamine residue was selected to be compared. Westernanalysis indicated that the variant VAVYKR (SEQ ID NO: 34) produced morelight chain than any other variant. VAVTKR (SEQ ID NO: 28) and VAVDKR(SEQ ID NO: 32) variants had more fusion protein indicating lessefficient cleavage. (FIG. 5).

To generate amino acid changes at the first Valine residue of the KEX2pre-sequence site (VAVEKR, SEQ ID NO: 9), a degenerate primer(GGACTAGTNNSGCCGTCGAGAAGCGCGACATCCAGATGACCCAG AG; SEQ ID NO:17) wassynthesized which was used in a PCR reaction with reverse primerCTAAAGGGAACAAAAGCTGGAGC (SEQ ID NO:14) to amplify DNA to generate PCRfragment with multiple sequences. The mixed PCR fragments were clonedinto Trichoderma expression vector (pTrex4). 30 clones were sequencedand 13 variants (MAVEKR (SEQ ID NO: 36), GAVEKR (SEQ ID NO: 37), AAVEKR(SEQ ID NO:38), LAVEKR (SEQ ID NO: 39), WAVEKR (SEQ ID NO: 40), KAVEKR(SEQ ID NO: 41), PAVEKR (SEQ ID NO: 42), RAVEKR (SEQ ID NO: 43), NAVEKR(SEQ ID NO: 44), TAVEKR (SEQ ID NO: 45), SAVEKR (SEQ ID NO: 46), QAVEKR(SEQ ID NO: 47) and EAVEKR (SEQ ID NO: 48)) were produced A new primerwas designed, synthesized(GGACTAGTNWCGCCGTCGAGAAGCGCGACATCCAGATGACCCAGAG SEQ ID NO:18) and usedin a PCR reaction with reverse primer CTAAAGGGAACAAAAGCTGGAGC (SEQ IDNO:14) to amplify DNA to generate PCR fragment with multiple sequences.The mixed PCR fragments were cloned into Trichoderma expression vector(pTrex4). 19 clones were sequenced and 5 more variants (YAVEKR (SEQ IDNO: 49), FAVEKR (SEQ ID NO: 50), DAVEKR (SEQ ID NO: 51), HAVEKR (SEQ IDNO: 52) and IAVEKR (SEQ ID NO: 53)) were produced. The plasmidscontaining the following 11 variants (MAVEKR (SEQ ID NO: 36), GAVEKR(SEQ ID NO: 37), AAVEKR (SEQ ID NO: 38), LAVEKR (SEQ ID NO:39), WAVEKR(SEQ ID NO: 40), KAVEKR (SEQ ID NO: 41), PAVEKR (SEQ ID NO: 42), HAVEKR(SEQ ID NO: 52), DAVEKR (SEQ ID NO: 51), SAVEKR (SEQ ID NO: 46) andQAVEKR (SEQ ID NO: 47)) were transformed biolistically into the T.reesei strain.

More than 20 transformants were obtained for each variant andtransferred to new plates. More than 8 stable transformants for eachvariant were selected and grown in Proflo media for 2 days at 28° C. 5mls of 2 days old culture from Proflo were transferred to 50 mls ofDefine media. The cultures were grown for 4 days at 28° C. Culturebroths were centrifuged and supernatants were analyzed by proteinSDS-PAGE. One transformant (the best producing transformant) from eachvariant was selected. Western analysis indicated (FIG. 6) that allvariants produced light chain. All showed less than 95% cleavage exceptLAVEKR (SEQ ID NO: 39). This variant showed more efficient KEX2 cleavagethan the VAVEKR (SEQ ID NO: 9) variant.

To generate amino acid changes at the Alanine residue of the KEX2 region(VAVEKR, (SEQ ID NO: 9)), a degenerate primer(GGACTAGTGTCNNSGTTGAGAAAGGCGACATCCAGATGACCCA GAGC; SEQ ID NO:19) wassynthesized which was used in a PCR reaction with reverse primer (SEQ IDNO:14) to amplify DNA to generate PCR fragment with multiple sequences.The mixed PCR fragments were cloned into Trichoderma expression vector(pTrex4). 96 clones were sequenced and 15 variants (VDVEKR (SEQ ID NO:54), VEVEKR (SEQ ID NO: 55), VGVEKR (SEQ ID NO: 56), VIVEKR (SEQ ID NO:57), VKVEKR (SEQ ID NO: 58), VLVEKR (SEQ ID NO: 59), VMVEKR (SEQ ID NO:60), VNVEKR (SEQ ID NO: 61), VPVEKR (SEQ ID NO:62), VRVEKR (SEQ ID NO:63), VSVEKR (SEQ ID NO: 64), VTVEKR (SEQ ID NO: 65), VVVEKR (SEQ ID NO:66), VWVEKR (SEQ ID NO: 67) and VYVEKR (SEQ ID NO: 68)) were produced. 5plasmids were transformed biolistically into the T. reesei strain. Morethan 20 transformants for each variant were obtained and transferred tonew plates. For this first set of 5 variants (VGVEKR (SEQ ID NO: 56),VTVEKR (SEQ ID NO: 65), VWVEKR (SEQ ID NO: 67), VEVEKR (SEQ ID NO: 55)and VPVEKR (SEQ ID NO: 62)), 10 stable transformants were selected. Forthe second set of 4 variants (VKVEKR (SEQ ID NO: 58), VRVEKR (SEQ ID NO:63), VVVEKR (SEQ ID NO: 66) and VIVEKR (SEQ ID NO: 57)), 10 stabletransformants were selected. The selected transformants were grown inProflo media for 2 days at 28° C. 5 mls of 2 days old culture fromProflo were transferred to 50 mls of Define media. The cultures weregrown for 4 days at 28° C. Culture broths were centrifuged andsupernatants are analyzed by protein SDS gel. One transformant (the bestproducing transformant) from each variant was selected to be compared(table 1). Western analysis (FIG. 7) indicated that only the free lightchain could be detected in the three variants: VGVEKR (SEQ ID NO: 56);VEVEKR (SEQ ID NO: 55) and VWVEKR (SEQ ID NO: 67). The variant VPVEKR(SEQ ID NO: 62) produced less free light chain and some uncleavedCBHI-light fusion.

To generate amino acid changes at the second valine residue of the KEX2site (VAVEKR, SEQ ID NO: 9), a degenerate primer(GGACTAGTGTCGCCNNSGAGAAACGCGACATCCAGATGACCCAG AG; SEQ ID NO:20) wassynthesized which was used in a PCR reaction with reverse primer (SEQ IDNO:14) to amplify DNA to generate PCR fragment with multiple sequences.The mixed PCR fragments were cloned into Trichoderma expression vector(pTrex4). 36 clones were sequenced and 15 variants (VAAEKR (SEQ ID NO:69), VADEKR (SEQ ID NO: 70), VAEEKR (SEQ ID NO: 71), VAFEKR (SEQ ID NO:72), VAGEKR (SEQ ID NO: 73), VAIEKR (SEQ ID NO: 74), VALEKR (SEQ ID NO:75), VANEKR (SEQ ID NO: 76), VAQEKR (SEQ ID NO: 77), VAREKR (SEQ ID NO:78), VASEKR (SEQ ID NO: 79), VATEKR (SEQ ID NO: 80), VAWEKR (SEQ ID NO:81), VAYEKR (SEQ ID NO: 82) and VAPEKR (SEQ ID NO: 83)) were produced.Plasmids were transformed biolistically into the T. reesei strain. Morethan 20 transformants for each variant were obtained and transferred tonew plates. For the first set of 8 variants (VAAEKR (SEQ ID NO: 69),VADEKR (SEQ ID NO: 70), VAEEKR (SEQ ID NO: 71), VAFEKR (SEQ ID NO: 72),VAGEKR (SEQ ID NO: 73), VANEKR (SEQ ID NO: 76), VALEKR (SEQ ID NO: 75)and VAIEKR (SEQ ID NO: 74)), 10 stable transformants were selected. Forthe second set of 2 variants (VASEKR (SEQ ID NO: 79) and VAREKR (SEQ IDNO: 78), 8 stable transformants were selected. Only 4 transformants wereselected for VAPEKR (SEQ ID NO: 83) variant. The selected transformantswere grown in Proflo media for 2 days at 28° C. 5 mls of 2 days oldculture from Proflo were transferred to 50 mls of Define media. Thecultures were grown for 4 days at 28° C. Culture broths were centrifugedand supernatants were analyzed. One transformant (the best producingtransformant) from each variant was selected to be compared (Table 1).Western analysis (FIG. 8) indicated that VAIEKR (SEQ ID NO: 74) andVALEKR (SEQ ID NO: 75) generated complete cleavage of the fusionpolypeptide since a fusion band was not observed in the gel. Westernblot (FIG. 8) indicated that VAFEKR (SEQ ID NO: 72) produced the highestamount of antibody light chain even though the cleavage was not 100%.

TABLE 1 MAVEKR VKVEKR VAAEKR VAVWKR (SEQ ID NO: 36) (SEQ ID NO: 58)(SEQ ID NO: 69) (SEQ ID NO: 25) GAVEKR VRVEKR VADEKR VAVGKR(SEQ ID NO: 37) (SEQ ID NO: 63) (SEQ ID NO: 70) (SEQ ID NO: 26) AAVEKRVVVEKR VAEEKR VAVRKR (SEQ ID NO: 38) (SEQ ID NO: 66) (SEQ ID NO: 71)(SEQ ID NO: 27) LAVEKR VIVEKR VAFEKR VAVTKR (SEQ ID NO: 39)(SEQ ID NO: 57) (SEQ ID NO: 72) (SEQ ID NO: 28) WAVEKR VEVE VAGEKRVAVVKR (SEQ ID NO: 40) (SEQ ID NO: 55) (SEQ ID NO: 73) (SEQ ID NO: 29)KAVEKR VGVEKR VAIEKR VAVAKR (SEQ ID NO: 41) (SEQ ID NO: 56)(SEQ ID NO: 74) (SEQ ID NO: 30) PAVEKR VPVEKR VALEKR VAVLKR(SEQ ID NO: 42) (SEQ ID NO: 62) (SEQ ID NO: 75) (SEQ ID NO: 31) SAVEKRVTVEKR VANEKR VAVDKR (SEQ ID NO: 46) (SEQ ID NO: 65) (SEQ ID NO: 76)(SEQ ID NO: 32) QAVEKR VWVEKR VASEKR VAVNKR (SEQ DI NO: 47)(SEQ ID NO: 67) (SEQ ID NO: 79) (SEQ ID NO: 33) DAVEKR VAREKR VAVYKR(SEQ ID NO: 51) (SEQ ID NO: 78) (SEQ ID NO: 34) HAVEKR VAPEKR VAVHKR(SEQ ID NO: 52) (SEQ ID NO: 83) (SEQ ID NO: 35)

Example 6 Construction of a Trastuzumab Light Chain Expression StrainContaining the NVISKR (SEQ ID NO: 22) KEX2 Region

A NVISKR KEX2 region is found naturally in the prosequence of the A.niger glucoamylase (glaA). To construct a fusion polypeptide an oligo,GGACTAGTAACGTCATCAGCAAGCGCGACATCCAGATGACCCAGAGC (SEQ ID NO. 21) wassynthesized by Invitrogen and used to amplify light chain DNA withreverse primer (SEQ ID NO. 14), The resulting PCR fragment encodes lightchain and NVISKR (SEQ ID NO:22) sequence for kex2 cleavage. The PCRfragment was digested with restriction enzymes SpeI and AscI and clonedto expression Vector pTrex4 to generate a plasmid named aspTrex4-NVIS-her2 light chain geneart (KR-TR). Fidelity of the PCRfragment was analyzed by DNA sequencing. The plasmid was transformedbiolistically into the Trichoderma reesei strain. More than 20transformants were obtained and transferred to new plates. 10 stabletransformants were selected to grow in Proflo media for 2 days at 30° C.5 mls of 2 days old culture from Proflo were transferred to 50 mls ofDefine media. The cultures were grown for 5 days at 28° C. Culturebroths were centrifuged and supernatants were used for protein analysis.Western analysis indicated that more than 95% of the fusion proteinswere cleaved (FIG. 6).

Example 7 Construction of a Trastuzumab Light Chain Expression StrainContaining the SDVTKR (SEQ ID NO: 24) KEX2 Region

An oligo, GGACTAGTAGCGACGTCACCAAGCGCGACATCCAGATGACCCAGAGC (SEQ ID NO:23) was synthesized by Invitrogen and used to amplify light chain DNAwith reverse primer (SEQ ID NO: 14), The resulting PCR fragment encodeslight chain and SDVTKR (SEQ ID NO: 24) sequence for kex2 cleavage. ThePCR fragment was digested with restriction enzymes SpeI and AscI andcloned to expression Vector pTrex4 to generate a plasmid named aspTrex4-SDVT-her2 light chain geneart (KR-TR). Fidelity of the PCRfragment was analyzed by DNA sequencing. The plasmid was transformedbiolistically into the Trichoderma reesei strain. More than 20transformants were obtained and transferred to new plates. 10 stabletransformants were selected to grow in Proflo media for 2 days at 30° C.5 mls of 2 days old culture from Proflo were transferred to 50 mls ofDefine media. The cultures were grown for 5 days at 28° C. Culturebroths were centrifuged and supernatants were used for protein analysis.Western analysis indicated that more than 50% of the fusion proteinswere cleaved (FIG. 6).

Example 8 Construction of a Trastuzumab Light Chain Expression StrainContaining the VAVEKR (SEQ ID NO: 9) KEX2 Region in Aspergillus niger

The plasmid (pTrex4-VAVE-her2 light chain geneart (KR-TR) from Example 5was digested with SpeI and AscI. The end of the DNA fragment of the AscIcutting site was blunted by T4 DNA polymerase. The fragment was isolatedon a 1.2% agarose gel and ligated to A. niger expression plasmid(pSLGAMpR2—BBI as disclosed in US Patent Publication No. 2005 0153399)which was cut with NheI and BstEII with the BstEII end blunted with T4DNA polymerase. The new plasmid, named pSLGAMpR2—VAVE-her2 LC geneartwas transformed into A. niger strain dgr246:Δamy5;pyr-which is derivedfrom the dgr246:ΔGAP:pyr-strain disclosed in US Pat. Pub. 20050153399.The difference being that the protein level of α-amylase is greatlyreduced in this plasmid because of a mutation.

The dgr246ΔGAP:pyr2- is derived from strain dgr246 P2 which has the pepAgene deleted, is pyrG minus and has undergone several rounds ofmutagenesis and screening or selection for improved production of aheterologous gene product (Ward, M. et al., 1993, Appl. Microbiol.Biotech. 39:738-743 and references therein). To create straindgr246AGAP:pyr2- the glaA (glucoamylase) gene was deleted in straindgr246 P2 using exactly the same deletion plasmid (pAGAM NB-Pyr) andprocedure as reported by Fowler, T. et al (1990) Curr. Genet.18:537-545. Briefly, the deletion was achieved by transformation with alinear DNA fragment having glaA flanking sequences at either end andwith part of the promoter and coding region of the glaA gene replaced bythe Aspergillus nidulans pyrG gene as selectable marker. Transformantsin which the linear fragment containing the glaA flanking sequences andthe pyrG gene had integrated at the chromosomal glaA locus wereidentified by Southern blot analysis. This change had occurred intransformed strain dgr246AGAP. Spores from this transformant were platedonto medium containing fluoroorotic acid and spontaneous resistantmutants were obtained as described by van Hartingsveldt, W. et al.(1987) Mol. Gen. Genet. 206:71-75. One of these, dgr246ΔGAP:pyr2-, wasshown to be a uridine auxotroph strain which could be complemented bytransformation with plasmids bearing a wild-type pyrG gene.

More than 20 transformants were obtained and transferred to new plates.17 transformants were grown in Promosoy medium for 5 days at 28° C.Culture broths were centrifuged and supernatants were used for proteinSDS PAGE and Western analysis. Data indicated that all transformantsproduced antibody light chain. The transformant #A12 produced the mostantibody light chain, and 60-70% of the fusion protein was cleaved (FIG.9).

The preceding description merely illustrates principles of exemplaryembodiments. It will be appreciated that those skilled in the art willbe able to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions.

Moreover, all statements herein reciting principles, aspects, andembodiments of the invention as well as specific examples thereof, areintended to encompass both structural and functional equivalentsthereof. Additionally, it is intended that such equivalents include bothcurrently known equivalents and equivalents developed in the future,i.e., any elements developed that perform the same function, regardlessof structure. The scope of the present invention, therefore, is notintended to be limited to the exemplary embodiments shown and describedherein.

1-30. (canceled)
 31. A method for cleaving a desired protein from arecombinant fusion polypeptide comprising expressing in a filamentousfungal cell a fusion polypeptide encoded by a fusion DNA constructencoding a fusion polypeptide comprising in operable linkage from the 5′end of said construct: a) a promoter, b) a first DNA molecule encoding asignal sequence, c) a second DNA molecule encoding a carrier protein, d)a third DNA molecule encoding a KEX2 region, said region comprising aKEX2 site (B₁B₂) and a KEX2 site pre-sequence ((X)n=2 to 6) immediately5′ to the KEX2 site, and e) a fourth DNA molecule encoding a desiredprotein; and wherein said KEX2 region provides a protein cleavage siteand obtaining a desired protein which is cleaved from the expressedfusion polypeptide.
 32. The method according to claim 31, wherein thedesired protein is a therapeutic protein.
 33. The method according toclaim 31, wherein the desired protein is an antibody.
 34. The methodaccording to claim 31, wherein the cleavage of the desired protein isincreased compared to the cleavage of said desired protein from anequivalent fusion polypeptide lacking the KEX2 site pre-sequence. 35.The method according to claim 26, wherein the KEX2 region is X₄X₃X₂X₁KRand X₄ is V and X₁ is E, S, T or Y.
 36. A method for increasing theproduction of an antibody from a filamentous fungal cell comprising: a)obtaining a filamentous fungal cell comprising a fusion DNA constructencoding a fusion polypeptide comprising in operable linkage from the 5′end of said construct: i) a promoter, ii) a first DNA molecule encodinga signal sequence, iii) a second DNA molecule encoding a carrierprotein, iv) a third DNA molecule encoding a KEX2 region X₄X₃X₂X₁KR,wherein X₄ is V, S, N, L, or K; X₃ is A, V, D, W, E or P; X₂ is V, I, Lor F; and X₁ is E, S, T or Y, and v) a fourth DNA molecule encoding adesired protein; b) culturing the fungal cell under suitable conditionsfor expression of the fusion polypeptide; and c) wallowing secretion ofthe fusion polypeptide, wherein the secretion of the desired protein isincreased compared to the secretion of an equivalent fusion polypeptidenot including a KEX2 pre-sequence.
 37. The method of claim 36, whereinthe secretion of the desired protein is increased by at least 30%compared to the secretion of the desired protein from the equivalentfusion polypeptide.
 38. (canceled)
 39. A method for identifying enhancedsecretion and/or cleavage of a desired protein comprising: a) altering aKEX2 site pre-sequence of a parental fusion polypeptide, said parentalfusion polypeptide comprising a signal sequence; a KEX2 regioncomprising a KEX2 site (B1B2) and a KEX2 site pre-sequence ((X)_(n)=4which is located immediately N-terminal to said KEX2 site, and an aminoacid sequence comprising a desired protein to produce a set of testrecombinant fusion polypeptides that are identical to said parentalfusion polypeptide except for said KEX2 site pre-sequence; b) evaluatingsecretion and/or cleavage of said test fusion polypeptides and saidparental fusion polypeptide by a filamentous fungal cell; and c)identifying a test fusion polypeptide that has enhanced secretion and/orcleavage as compared to said parental fusion polypeptide.
 40. The methodaccording to claim 39 further comprising identifying an optimized KEX2site pre-sequence which comprises, testing a plurality of different testfusion polypeptides, and determining which of said different test fusionpolypeptides has greater secretion and/or protein cleavage, wherein saidoptimized KEX2 site pre-sequence is the altered KEX2 site pre-sequenceof the test recombinant fusion polypeptide that has the greatestsecretion and/or protein cleavage.